Lack of interleukin-1α in Kupffer cells attenuates liver inflammation and expression of inflammatory cytokines in hypercholesterolaemic mice

Digestive and Liver Disease 46 (2014) 433–439

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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld

Liver, Pancreas and Biliary Tract

Lack of interleukin-1␣ in Kupffer cells attenuates liver inflammation and expression of inflammatory cytokines in hypercholesterolaemic mice Sarita Olteanu a,1 , Michal Kandel-Kfir a,1 , Aviv Shaish a , Tal Almog a , Shay Shemesh a , Iris Barshack b,c , Ron N. Apte d , Dror Harats a,c , Yehuda Kamari a,c,∗ a

The Bert W. Strassburger Lipid Center, Sheba Medical Center, Tel Hashomer, Israel Pathology Department, Sheba Medical Center, Tel Hashomer, Israel c Sackler Faculty of Medicine, Tel-Aviv University, Israel d Department of Microbiology and Immunology, Ben Gurion University, Beer Sheva, Israel b

a r t i c l e

i n f o

Article history: Received 28 November 2013 Accepted 20 January 2014 Available online 26 February 2014 Keywords: Cytokine Fatty liver IL-1 Inflammation Kupffer cells NASH

a b s t r a c t Background: The role of Kupffer cell interleukin (IL)-1 in non-alcoholic steatohepatitis development remains unclear. Aims: To evaluate the role of Kupffer cell IL-1␣, IL-1␤ or IL-1 receptor type-1 (IL-1R1) in steatohepatitis. Methods: C57BL/6 mice were irradiated and transplanted with bone marrow-derived cells from WT, IL1␣−/−, IL-1␤−/− or IL-1R1−/− mice combined with Kupffer cell ablation with Gadolinium Chloride, and fed atherogenic diet. Plasma and liver triglycerides and cholesterol, serum alanine aminotransferase (ALT), liver histology and expression levels of inflammatory genes were assessed. Results: The ablation and replacement of Kupffer cells with bone marrow-derived cells was confirmed. The atherogenic diet elevated plasma and liver cholesterol, reduced plasma and liver triglycerides and increased serum ALT levels in all groups. Steatosis and steatohepatitis were induced, but without liver fibrosis. A reduction in the severity of portal inflammation was observed only in mice with Kupffer cell deficiency of IL-1␣. Accordingly, liver mRNA levels of inflammatory genes encoding for IL-1␣, IL-1␤, TNF␣, SAA1 and IL-6 were significantly lower in mice with Kupffer cell deficiency of IL-1␣ compared to WT mice. Conclusion: Selective deficiency of IL-1␣ in Kupffer cells reduces liver inflammation and expression of inflammatory cytokines, which may implicate Kupffer cell-derived IL-1␣ in steatohepatitis development. © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

1. Introduction Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver disease throughout the world [1]. It reflects a large spectrum of liver diseases ranging from benign fatty liver (steatosis) to non-alcoholic steatohepatitis (NASH), which can lead to cirrhosis and liver cancer [2]. NASH is accompanied by histological changes of fat accumulation, hepatocellular injury, inflammation (hepatitis) and scarring (fibrosis). The pathogenesis of NASH involves various hits, including lipotoxicity, gut-derived signals such as endotoxin, signals from the innate immune system such as toll like receptors (TLRs) or pro-inflammatory cytokines, oxidative and endoplasmic reticulum

∗ Corresponding author at: The Bert Strassburger Lipid Center, Sheba Medical Center, Tel Hashomer 52621, Israel. Tel.: +972 3 5302940; fax: +972 3 5343521. E-mail address: [email protected] (Y. Kamari). 1 Both are first authors.

stress [3]. The lipotoxic molecules implicated in the pathogenesis of NASH have not been fully clarified, and currently, most in favour are non-triglyceride lipids such as free fatty acids and free cholesterol [4,5]. Hepatocyte apoptosis due to lipotoxicity and release of inflammatory content of secondary necrotic apoptotic cells initiate sterile inflammation in the liver [6,7]. Kupffer cells, the liver-resident macrophages, release a broad panel of inflammatory cytokines such as TNF␣, IL-6 and IL-1 [3]. IL-1␣ and IL-1␤ induce the expression of a variety of pro-inflammatory genes upon binding to IL-1 receptor type 1 (IL-1R1) [8,9]. Both IL-1␣ and IL-1␤ are synthesized as precursors (31 kDa) and are processed to mature forms (17 kDa) via specific cellular proteases [8,10]. IL-1␤ is generated upon inflammatory signals and is only active as a mature secreted protein after cleavage by caspase-1. In contrast, IL-1␣ exerts its effects both in the mature and the precursor forms when binding to IL-1R1 [8,10,11]. IL-1␣ belongs to a newly recognized group of dual-function cytokines that play a role in the nucleus apart from their extracellular, receptor-mediated effects [15,16].

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S. Olteanu et al. / Digestive and Liver Disease 46 (2014) 433–439

When released from dying cells, IL-1␣ initiates sterile inflammation, acting as a damage-associated molecular pattern molecule [8,13]. The link between Kupffer cell-derived IL-1␤ and regulation of hepatic lipid accumulation has been recently suggested. Kupffer cells are the main source of IL-1␤ and depletion of Kupffer cells reduced IL-1␤ expression and liver steatosis under high fat diet [10,12,13]. Activation of Kupffer cells through TLR-4 promotes NASH by producing inflammatory cytokines, including TNF␣ and IL-1␤ [13] and IL-1R1 deficiency attenuates steatohepatitis [12]. However, the contribution of Kupffer cell-derived IL-1␤ and IL-1␣ to the development of NASH has not been directly addressed. We recently reported that both IL-1␣ and IL-1␤-deficient mice were almost entirely protected from atherogenic diet-induced NASH and liver fibrosis, suggesting that these cytokines might be crucially involved in the development of liver inflammation in the context of fatty liver [14]. In the present study we sought to replace the resident Kupffer cells with gene-targeted bone marrow (BM)-derived cells in order to examine whether deficiency of IL-1␣, IL-1␤ or IL1RI in Kupffer cells protects against atherogenic diet-induced NASH and liver fibrosis. Our results suggest that Kupffer cell-derived IL1␣ and not IL-1␤ contributes to the inflammatory milieu of fatty liver disease in mice. 2. Materials and methods 2.1. Animals and experimental procedures The generation of IL-1␣, IL-1␤ and IL-1RI KO mice, has been described previously [15,16]. WT C57BL/6 mice were purchased from Harlan, Israel. Mice were maintained on a 12-h light/12-h dark cycle and fed a chow diet containing 4.5% fat by weight (0.02% cholesterol) (TD19519: Koffolk, Israel). The studies were carried out according to the protocols approved by the Sheba Medical Center Board for Studies in Experimental Animals. To induce fatty liver, mice were fed the atherogenic diet containing 17% total fat, 1.25% cholesterol and 0.5% sodium-cholate (Teklad Premier Laboratory Diet No. TD 90221). To study whether deficiency of IL-1␣, IL-1␤ or IL-1RI in Kupffer cells protects against NASH development, we created the different transplanted groups. We used 10-week-old male WT C57BL6/J mice as recipients and female WT C57BL6/J, IL-1␣−/− , IL-1␤−/− and IL-1RI−/− mice as donors. The experimental groups: WT cells transplanted to WT mice fed with chow diet (WT → WT chow) and atherogenic diet-fed WT → WT, IL-1␣−/− cells to WT (IL-1␣−/− → WT), IL-1␤−/− cells to WT (IL-1␤−/− → WT) and IL-1RI−/− cells to WT mice (IL1RI−/− → WT). 2.2. Bone marrow transplantation (BMT) and Kupffer cell ablation Ten-week-old male WT C57BL/6 mice underwent the bone marrow transplantation procedure as previously described [14]. Mice were fed with a standard chow diet for 5 weeks. When using the BMT procedure in mice it is estimated that the majority of Kupffer cells are replaced after a recovery period of 9 weeks [17]. Moreover, chemical depletion of Kupffer cells speeds up the replacement of host cells by BM derived cells [18]. To ensure replacement of host cells, we used Gadolinium Chloride (GC) for Kupffer cell ablation; Five weeks after the BMT procedure, mice were injected (tail vein) with saline containing GC (25 mg/kg in saline, 0.2 mL) (Cat# 439770, Lot# 16169EJ, Sigma–Aldrich) and fed standard chow diet for additional 5 weeks. 2.3. Chemical analysis of serum and tissue Animals were sacrificed by inhaling CO2 and blood was collected after a 16-h fast, by a puncture of the inferior vena

cava. In some experiments, blood was obtained from a puncture of the retro orbital sinus under mild isoflurane anaesthesia. Livers were immediately collected and weighed, and tissue samples were frozen in liquid nitrogen and stored at −80 ◦ C. Serum analysis of ALT levels was performed using an automated enzymatic technique (Boehringer Mannheim). Total plasma cholesterol and TG levels were determined using an automated enzymatic technique (Boehringer Mannheim, Germany). Hepatic lipids were extracted according to the method of Folch et al. [19]. The extract was dissolved in 2-propanol and subsequently analyzed for total cholesterol, and TGs using an automated enzymatic technique (Boehringer Mannheim, Germany). 2.4. Tissue preparation and histology Livers were harvested, fixed overnight in 4% paraformaldehyde then embedded in paraffin and sectioned (6-␮m thickness). All samples were routinely stained for general morphology with haematoxylin-eosin and for collagen with Masson’s trichrome. The histopathological score was performed in a blinded fashion. 2.5. Analysis of gene expression by real-time PCR Total RNA was isolated from frozen tissues, treated with DNase and purified using the Qiagen Rneasy Mini Kit, following the manufacturer’s protocol (Qiagen, Hilden, Germany). RNA was reversetranscribed using the High-Capacity cDNA Reverse Transcription Kits according to manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). Real time PCR was carried out using a 7500 sequence detection System (Applied Biosystems). Gene expression profiling was achieved using the comparative cycle threshold (ct) method of relative quantification using Glyceraldehyde-3phosphate dehydrogenase (GAPDH) as the endogenous gene. Analysis of real-time PCR data was done using the Ct method. PCR primers for Taqman/Probe Library assays (Appendix A) were designed with the Probe Library Assay Design Center (http://www. roche-applied-science.com/sis/rtpcr/upl/adc.jsp). 2.6. Statistical analyses The values reported are the mean ± SE. Student’s t-test was used, where applicable, and an ANOVA analysis was applied to assess interactions between groups and differences between means. P < 0.05 is accepted as statistically significant. 3. Results We first established the experimental mouse model of replacing Kupffer cells with transplanted cells, by using the BMT procedure combined with Kupffer cell ablation (Figs. S1–S5). We used this model to study whether deficiency of IL-1␣, IL-1␤ or IL-1RI in Kupffer cells protects from the development of NASH. After irradiation, 10-week-old male WT C57BL6/J mice were transplanted with BM-derived cells from WT (WT → WT), IL-1␣−/− (IL-1␣−/− → WT), IL-1␤−/− (IL-1␤−/− → WT) and IL-1RI−/− (IL-1RI−/− → WT) mice. Following the BMT and Kupffer cell ablation procedures, the atherogenic diet was given to the mice for 11 weeks. A control group of WT → WT mice was fed chow diet. 3.1. Serum ALT levels and total plasma cholesterol and TGs in chow-fed mice We measured serum ALT as a parameter of liver injury and total plasma cholesterol and TG levels, 10 weeks after the BMT procedure prior to atherogenic diet feeding. Serum ALT and plasma cholesterol and TG levels were similar in all groups (Fig. 1A–C).

S. Olteanu et al. / Digestive and Liver Disease 46 (2014) 433–439

A

80

ALT (units/L)

60

40

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IL

160 140 120 100 60

3.3. mRNA levels of inflammatory cytokines

40 20

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WT → WT chow-fed mice and plasma cholesterol was higher in IL-1␣−/− → WT compared to WT → WT fed the atherogenic diet (Fig. 2). Liver cholesterol and TG content was similar in IL-1␣−/− → WT compared to WT → WT fed the atherogenic diet (Fig. 2). We assessed the severity of NASH in this experimental model with a histopathology score for steatosis, liver inflammation and fibrosis. The morphology of livers from WT → WT mice following the atherogenic diet had been dramatically altered compared to WT → WT on chow diet with extensive parenchymal steatosis (about 90% of the lobule) and moderate to severe lobular and portal inflammation (Fig. 3). These morphological changes are compatible with severe steatohepatitis. However, in this experimental model, severe steatosis and inflammation were not accompanied by liver fibrosis. This is in contrast to WT mice given atherogenic diet without the BMT and ablation procedures, which develop severe NASH with extensive fibrosis. The degree of steatosis was similar in all the atherogenic diet-fed groups (Fig. 3B and Table 1). The degree of lobular and portal inflammation after the atherogenic diet was similar in IL-1␤−/− → WT and IL-1RI−/− → WT compared to WT → WT, whereas IL-1␣−/− → WT mice exhibited a reduction in severity of portal inflammation (Fig. 3B and Table 1).

80

W

C

-t

to -1 R

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Plasma cholesterol (mg/dl)

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a/-

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100 80 60 40 20

T W -t o

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-1 R

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-/-1 b IL

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to T W

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ow ch T W to T W

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Fig. 1. IL-1␣, IL-1␤ or IL-1RI deficiency in Kupffer cells does not affect serum ALT levels and total plasma cholesterol and TGs in mice fed chow diet. Serum ALT (A), plasma cholesterol (B) and TG (C) were measured in mice fed chow diet following the BMT and ablation procedure. WT → WT chow (n = 7), WT → WT (n = 7), IL-1␣−/− → WT (n = 7), IL-1␤−/− → WT (n = 7) and IL-1RI−/− → WT (n = 7). Blood was obtained from fasted animals. Data represent mean ± SE of each group.

3.2. Liver inflammation All the experimental groups given the atherogenic diet for 11 weeks developed fatty liver, manifested by increased liver to body weight ratio, increased liver and plasma cholesterol, increased serum ALT, and decreased plasma TG, compared to WT → WT on chow diet (Fig. 2). Liver weight was slightly higher in IL-1␤−/− → WT compared to IL-1␣−/− → WT mice with no difference in liver TGs and cholesterol, serum ALT and plasma TGs in all the atherogenic diet-fed groups (Fig. 2). Liver TGs were lower in IL-1␤−/− → WT and IL-1RI−/− → WT compared to

We assessed the expression of inflammation and liver fibrosisrelated genes in livers from WT → WT fed chow diet and WT → WT, IL-1␣−/− → WT, IL-1␤−/− → WT and IL-1RI−/− → WT mice that were fed atherogenic diet for 11 weeks. First, compared to chow-fed, WT → WT mice fed atherogenic diet showed a 2.7, 4.3, 25.5 and 2.8 fold increases in mRNA transcript levels of TNF␣, the acute phase proteins SAA1, IL-1Ra and the macrophage marker CD68, respectively (Fig. 4A–D). There was also a 1.7 and 5.4 fold increase in mRNA levels of TGF␤ and procollagen type I alpha1 (Col1a1) (Fig. 4E and F). The mRNA levels of IL-1␣ (Fig. 4G) and IL-6 (Fig. 4I) were similar in WT → WT mice on atherogenic diet compared to WT → WT fed chow diet, and mRNA levels of IL-1␤ were about 2 fold lower in WT → WT on atherogenic diet (Fig. 4H). The IL-1␤ mRNA levels were undetectable in IL-1␤−/− → WT mice, indicating that Kupffer cells are the primary source of IL-1␤ in this experimental model and that the BMT and ablation procedure was successful. The expression of IL-1␣ in livers of IL-1␣−/− → WT mice was reduced by 60% compared to WT → WT on atherogenic diet, suggesting that other cell types besides Kupffer cells and bone marrow-derived cells also produce IL-1␣ in this model (Fig. 4G). In addition, the expression of IL-1␣ was unaffected in IL-1␤−/− → WT compared to WT → WT mice fed atherogenic diet (Fig. 4G), indicating no regulatory effect of IL-1␤ on expression of IL-1␣ in bone marrow derived cells. IL1␤−/− → WT mice did not show reduced expression of TNF␣, IL-6, SAA1 or TGF␤ and in fact IL-1Ra, CD68 and collagen were 2.2, 1.5 and 1.9 fold higher compared to WT → WT mice on atherogenic diet (Fig. 4C, D and F). In IL-1RI−/− → WT mice, the mRNA levels of SAA1, IL-1Ra, collagen. IL-1␣, IL-1␤ and IL-6 were similar compared to WT → WT mice (Fig. 4B, C, F, G, H and I) whereas TNF␣ and TGF␤ were 46 and 45% lower (Fig. 4A and E). IL-1␣ deficiency in Kupffer cells had the most effect on expression of inflammatory genes in the liver with 50%, 68% and 41% reduction in the expression of TNF␣, SAA1 andIL-6, respectively compared to WT → WT mice (Fig. 4A, B and I). In addition, IL-1␤ mRNA levels were 67% lower in IL-1␣−/− → WT mice compared to WT → WT mice (Fig. 4H) suggesting the possible regulation of IL-1␤ expression by IL-1␣. The mRNA levels of TGF␤ and collagen were similar in IL-1␣−/− → WT compared to WT → WT mice on atherogenic diet. To summarize, IL-1␣−/− → WT mice uniquely showed a significant and consistent down regulation of inflammatory genes following feeding with the atherogenic diet compared to WT → WT mice.

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A

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Liver cholesterol (mg/g)

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1

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5

Fig. 2. IL-1␣, IL-1␤ or IL-1RI deficiency in Kupffer does not affect the degree of fatty liver formation and serum ALT levels. Liver to body weight ratio (A), liver cholesterol (B), liver TG (C), (WT → WT chow (n = 6), WT → WT (n = 8), IL-1␣−/− → WT (n = 10), IL-1␤−/− → WT (n = 9) and IL-1RI−/− → WT (n = 8)), serum ALT (D), plasma cholesterol (E) and plasma TG (F) (WT → WT chow (n = 6), WT → WT (n = 17), IL-1␣−/− → WT (n = 19), IL-1␤−/− → WT (n = 12) and IL-1RI−/− → WT (n = 15)) levels were measured after 11 weeks of atherogenic diet. Blood was obtained from fasted animals. Data represent mean ± SE of each group (a,b P < 0.05 compared to all other groups, **P < 0.05 between selected groups).

4. Discussion The aim of the study was to assess the role of Kupffer cell IL1 and IL-1R1, in fatty liver disease. We used the bone marrow transplantation procedure combined with Kupffer cell ablation to

generate mice with selective deficiency of IL-1␣, IL-1␤ or IL-1R1 in myeloid cells including Kupffer cells. The atherogenic diet induced steatosis and steatohepatitis but did not induce liver fibrosis. Our data show that Kupffer cell deficiency of IL-1␣, IL-1␤ or IL-1RI did not affect the extent of hepatic lipid accumulation. Histology score

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437

Table 1 Haematoxylin and eosin histopathology score.

Steatosis parenchymal involvement Fibrosis Inflammation Lobular Portal Liver cell injury Ballooning Acidophil bodies

WT to WT

WT to WT

Chow

Atherogenic

IL-1␣−/− to WT

IL-1␤−/− to WT

IL-1R−/− to WT

Scale

0.3 ± 0.2 0.0 ± 0.0

3.0 ± 0.0 0.0 ± 0.0

3.0 ± 0.0 0.0 ± 0.0

2.9 ± 0.1 0.0 ± 0.0

3.0 ± 0.0 0.0 ± 0.0

0–3 0–4

1.0 ± 0.3 0.8 ± 0.3

2.4 ± 0.2 2.8 ± 0.2

1.9 ± 0.2 1.8 ± 0.3*

2.3 ± 0.3 2.0 ± 0.2

2.7 ± 0.2 2.8 ± 0.2

0–3 0–3

0.0 ± 0.0 0.0 ± 0.0

1.4 ± 0.2 0.9 ± 0.0

1.7 ± 0.2 0.9 ± 0.1

1.8 ± 0.2 1.0 ± 0.0

1.5 ± 0.2 0.9 ± 0.1

0–2 0–1

Key: WT, wild-type; steatosis parenchymal involvement (<5% 0, 5–33% 1, 33–66% 2, >66% 3); fibrosis (none 0, perisinusoidal/periportal 1, perisinusoidal and periportal 2, bridging fibrosis 3, cirrhosis 4); portal inflammation (none/minimal 0, mild 1, moderate 2, severe 3); lobular inflammation (none 0, mild 1, moderate 2, and severe 3); liver cell injury: ballooning (none 0, few 1, many 2); acidophil bodies (none/rare 0, many 1). * P < 0.05 compared to “WT to WT”.

Fig. 3. Histological score of fat accumulation and inflammation. Representative macroscopic pictures of livers from WT → WT chow, WT → WT, IL-1␣−/− → WT, IL-1␤−/− → WT and IL-1RI−/− → WT mice (A). Liver sections were prepared for histology and stained with haematoxylin and eosin. Magnification 200× (B). Circle indicating fat accumulation in hepatocytes and arrow indicates inflammatory cells.

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Fig. 4. Gene expression analysis of inflammation and fibrosis related genes. Quantitative real-time PCR analysis of mRNA levels in liver samples of WT → WT on chow diet (n = 6) and WT → WT (n = 9), IL-1␣−/− → WT (n = 10), IL-1␤−/− → WT (n = 9) and IL-1RI−/− → WT (n = 7) mice after 11 weeks of atherogenic diet. Data represent mean ± SE. For statistical analysis, t-test between the specified groups (WT → WT chow compared to WT → WT samples (*P < 0.05) and IL-1␣−/− → WT, IL-1␤−/− → WT and IL-1RI−/− → WT compared to WT → WT samples (**P < 0.05)) was performed and a P-value was calculated (selected gene/GAPDH).

analysis revealed a reduction in portal inflammation in mice with Kupffer cell deficiency of IL-1␣. In line with the histology findings, liver mRNA levels of the inflammatory genes encoding for IL-1␣, IL-1␤, TNF␣, SAA1 and IL-6 were significantly lower in mice with selective deficiency of IL-1␣ in Kupffer cells compared to WT mice. In addition to activation of inflammatory responses in the liver, cytokine signalling pathways have also been reported to increase liver lipid accumulation which further amplifies the fat-induced inflammatory response [20–22]. Yet, very little is known about the role of IL-1␣ in lipid metabolism and the role of Kupffer cellsderived IL-1␣ in lipid metabolism and fatty liver has not been studied. Previous studies in our laboratory employing IL-1␣ KO mice showed that deficiency of IL-1␣ resulted in slightly more cholesterol accumulation during the steatosis phase [14]. In the present study, accumulation of liver cholesterol was not affected by Kupffer cell IL-1␣ deficiency suggesting that IL-1␣ derived from other hepatic cell types (hepatocytes or stellate cells) plays a role in liver cholesterol accumulation. The role of Kupffer cell IL-1␤ in fatty liver has been investigated in a few studies. Deficiency of IL-1Ra which was accompanied by increased gene expression of IL-1␤ in the liver, deteriorated cholesterol metabolism and fatty liver under atherogenic diet, suggesting that increased IL-1␤ signalling mediated the deterioration of lipid metabolism and liver inflammation [14]. In line with this, a few more recent reports showed that IL-1␤ signalling aggravates accumulation of cholesterol [22] and TGs [20]. Miura et al. examined IL-1␤ production in Kupffer cells in the choline-deficient amino acid (CDAA) dietary model of steatohepatitis [12]. Intravenous injection of liposomal clodronate, which exclusively depleted Kupffer cells, reduced IL-1␤ mRNA levels. In addition, mRNA analysis of isolated hepatocytes, Kupffer cells, HSCs and sinusoidal endothelial cells from WT mice fed the CDAA diet, showed high levels of IL-1␤ only in the Kupffer cell fraction indicating that this cell type is the main source of IL1␤ in experimental steatohepatitis [12]. Cell culture experiments showed that IL-1␤ that binds to IL-1RI increases lipid accumulation in hepatocytes. In line with the in vitro results, IL-1RI deficient mice exhibited reduced steatosis indicating that IL-1R signalling contributes to steatosis development [12]. Kupffer cell depletion was associated in another study, with decreased expression of IL-1␤ and reduced liver steatosis via IL-1␤-dependent inhibition of PPAR␣ expression and activity [20]. We found that IL-1␤-deficient mice accumulate similar liver cholesterol levels as WT mice at the steatosis stage and are protected from steatohepatitis and liver fibrosis. At the steatohepatitis phase, IL-1␤-deficient mice had significantly reduced inflammation and also less steatosis compared to WT mice [14]. This suggests that inflammation through IL-1␤ promotes further accumulation of cholesterol in the liver which contributes to steatohepatitis and liver fibrosis development. Although these data support a possible scenario where Kupffer cell-derived IL-1␤ binds to IL-1RI in hepatocytes and promotes hepatic lipid accumulation, the results of the present study show that deficiency of IL-1␤ or IL-1RI in Kupffer cells does not affect the degree of hepatic lipid accumulation. We previously found that deficiency of IL-1␣ or IL-1␤ protects mice from developing atherogenic diet-induced severe NASH [14]. Bone marrow transplantation experiments revealed that IL-1␣ or IL-1␤ from parenchymal liver cells, rather than BM-derived cells, are critical for development of steatohepatitis and liver fibrosis in hypercholesterolaemic mice [14]. Because the atherogenic diet was administered only 2 weeks after the bone marrow transplantation procedure, our previous experiments could not distinguish whether the protective effect of IL-1 deficiency was due to lack of its expression in Kupffer cells or in hepatocytes. In the current work, to ensure complete repopulation of Kupffer cells with the transplanted cells, the experimental model included BMT, Kupffer cell

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ablation and a recovery period of 10 weeks before administration of atherogenic diet. Kupffer cell deficiency of IL-1␣ and not IL-1␤ or IL-1RI, resulted in a reduction of portal inflammation raising the possibility that Kupffer cell-derived IL-1␣ promotes steatohepatitis by acting in a paracrine fashion on IL-1RI in other liver cell types. Kupffer cell IL-1␣ deficiency also lowered liver mRNA levels of IL-1␣, IL-1␤, TNF␣, IL-6 and SAA1. In contrast, Kupffer cell IL-1␤ deficiency did not attenuate the expression of inflammatory genes, and Kupffer cell IL-1RI deficiency only reduced mRNA levels of TNF␣. These results suggest that the reduced expression of IL-1␤, IL-6 and SAA due to Kupffer cell IL-1␣ deficiency is not mediated by TNF␣. Previous work has shown that IL-1 upregulates the expression of acute phase proteins and inflammatory cytokines including IL-6 [23,24]. Moreover, these results emphasize the central role of Kupffer cell IL-1␣ in regulation of inflammatory pathways that promote the progression of steatosis to steatohepatitis. Kupffer cell IL-1␣ deficiency decreased liver mRNA levels of IL-1␤ whereas Kupffer cell deficiency of IL-1␤ did not affect mRNA levels of IL1␣. Importantly, Kupffer cell deficiency of IL-1RI did not reduce mRNA levels of IL-1␤. These in vivo results are in line with previous reports in cultured macrophages, showing the possible regulation of IL-1␤ transcription by IL-1␣ independent of IL-1R1 [25,26]. The complex use of irradiation, bone marrow transplantation, Kupffer cell ablation and atherogenic diet altered a few features of the steatohepatitis model. For instance, liver mRNA levels of IL-1␣ were not elevated and IL-1␤ mRNA levels were lower, in atherogenic versus chow-fed WT mice. Despite the lack of increase in liver IL-1 mRNA levels, liver inflammation was attenuated in mice with Kupffer cell IL-1␣ deficiency suggesting that even basal levels of IL-1␣ in Kupffer cells are sufficient to affect liver inflammation. In addition, the development of liver inflammation was not accompanied by liver fibrosis. This can possibly be explained by the lack of increase in IL-1␣. Alternatively, the use of GC for Kupffer cell ablation possibly reduced the capability of hepatic stellate cells to produce fibrotic factors. In addition, although the combined BMT and ablation enabled the repopulation of Kupffer cells with the transplanted myeloid cells, it is not clear that these cells have the same characteristics as the replaced Kupffer cells. In order to answer these questions, we are in the process of generating mice with specific deficiency of IL-1␣ in myeloid cells including Kupffer cells, by using the Cre-lox system. In conclusion, by using the BMT combined with GC-Kupffer ablation, we demonstrated that Kupffer cell IL-1␣ contributes to the transformation of steatosis to steatohepatitis possibly by binding to IL-1RI on other liver cell-types and thereby promoting liver inflammation. Conflict of interest None declared. Acknowledgments This study was supported in part by the Talpiot Sheba Medical Leadership grant (Yehuda Kamari). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dld.2014.01.156.

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