The inhibition of NLRP3 signaling attenuates liver injury in an α-galactosylceramide-induced hepatitis model

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Title

The inhibition of NLRP3 signaling attenuates liver injury in an α

_{-galactosylceramide-induced hepatitis model( 本文(Fulltext) )}

Author(s)

神戸, 歩

Report No.(Doctoral

Degree)

博士(医学) 医博甲第1123号

Issue Date

2020-03-25

Type

博士論文

Version

ETD

URL

http://hdl.handle.net/20.500.12099/79317

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

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The inhibition of NLRP3 signaling attenuates liver injury in an

a

-galactosylceramide-induced hepatitis model

Ayumu Kanbe

a

, Hiroyasu Ito

a,*

, Yukari Omori

a

, Akira Hara

b

, Mitsuru Seishima

a a_{Department of Informative Clinical Medicine, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu City 501-1194, Japan} b_{Department of Tumor Pathology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu City 501-1194, Japan}

a r t i c l e i n f o

Article history: Received 6 June 2017 Accepted 12 June 2017 Available online 14 June 2017 Keywords:

Immune-mediated liver injury Inﬂammasomes

NLRP3

Alpha-galactosylceramide

a b s t r a c t

Inflammasomes are involved in innate immune responses. Several NOD-Like receptors (NLRs) participate in the formation of inflammasomes. NACHT, LRR and PYD domains-containing protein 3 (NALP3) belongs to the NLR family and recognizes adenosine triphosphate (ATP), crystals, and Reactive Oxygen Species (ROS). This study examined the effect of inflammasomes on alpha-galactosylceramide (GalCer)-induced liver injury using NALP3-knockout (KO) mice. GalCer administration induced inflammasome activation and IL-1b-maturation. In NALP3-KO mice treated with GalCer, serum ALT levels were significantly reduced compared with those in GalCer-treated WT mice. Histological examination revealed decreased necrosis in NALP3-KO mice compared with WT mice, consistent with ALT levels. Expression of proin-flammatory cytokines (such as IL-6, and TNF-a) and chemokines was also significantly suppressed in NALP3-KO mice. Moreover,flow cytometry analysis revealed fewer infiltrating immune cells in the livers of GalCer-treated NALP3-KO mice. Inportantly, the frequency of MDSCs (CD11bþGr-1int_{cells), which}

suppress the immune response, was signiﬁcantly increased in GalCer-treated NALP3-KO mice. In conclusion, NALP3 inhibition attenuated liver injury in GalCer-induced hepatitis. The inhibition of NALP3 signaling coused be a therapeutic target in immune-mediated liver injury.

1. Introduction

Autoimmune hepatitis (AIH) is an immune-mediated in ﬂam-mation and liver injury of uncertain cause. It is characterized by an inﬁltration of immune cells in the liver, an increase in

g

-globulins, antiantibodies, and HLA associations. Immunosuppressive therapy such as treatment with corticosteroids is effective for AIH [1]. Concanavalin A (Con A) has been widely used to model host immune-mediated liver injury [2]. Con A administration in vivo induces lymphocyte activation and recruitment into the liver. The Con A-induced hepatitis model has a unique pathogenesis as well as characteristics of immune-mediated hepatitis in humans, including autoimmune hepatitis and acute viral hepatitis. Previous studies demonstrated that natural killer T (NKT) cells are involved in the development of Con A-induced hepatitis model. Deficiency of NKT cells significantly reduced Con A-induced liver injury [3]. Activation of NKT cells by alpha-galactosylceramide (GalCer) also induces liver inflammation and injury [4,5]. Thus, AIH can be

studied in an immune-mediated liver injury model using Con A and GalCer.

The innate immune system is activated when pattern recogni-tion receptors (PRRs) detect a pathogen. The principle PRRs families are the Toll-like receptors (TLRs), the NOD-Like receptors (NLRs), and the RIG-I-Like receptors (RLRs) [6]. NLRs are intracellular sensors of pathogen-associated molecular patterns (PAMPS), and several NLRs are involved in the formation of inﬂammasomes[7]. The formation of inﬂammasome-related molecules leads to the activation of caspase-1, resulting in the cleavage of pro-IL-1

b

and pro-IL-18 into their mature forms. IL-1

b

and IL-18 are critical for the progression of inflammation in the microenvironment. Several re-ports have examined the role of in_{flammasomes in auto-immune} diseases. A recent study found that inflammasome activation was involved in the pathogenesis of collagen-induced arthritis [8]. NACHT, LRR and PYD domain-containing protein 3 (NALP3) plays a critical role in the development of systemic lupus erythematosus (SLE)[9]. Moreover, many reports indicate that inflammasome ac-tivity is related to auto-immune diseases in humans [10e12]. However, it remains unclear whether the activity of in flamma-somes is involved in AIH, which is one of the immune-mediated

* Corresponding author.

E-mail address:[email protected](H. Ito).

Contents lists available atScienceDirect

Biochemical and Biophysical Research Communications

j o u r n a l h o m e p a g e :w w w . e l s e v ie r . c o m / l o c a t e / y b b rc

http://dx.doi.org/10.1016/j.bbrc.2017.06.049

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diseases.

In this study, we used NALP3-KO mice to examined the role of NALP3, in the progression of GalCer-induced liver injury using NALP3-KO mice. We demonstrated that NALP3 deﬁciency reduced the inﬂammatory response in the liver and thus liver injury, by increasing CD11bþ/Gr-1intermediatecells.

2. Materials and methods 2.1. Mice

C57BL/6J (H-2b) wild-type (WT) mice (age, 6e10 weeks; weight; 20e30 g) were obtained from Japan SLC Inc. (Shizuoka, Japan). LRR and PYD domains-containing protein 3 (NALP3) knockout (KO) mice on a C57BL/6J background were obtained from Genentech, Inc. (South San Francisco, CA). All procedures were conducted in accordance with the guidelines of the National In-stitutes of Health Guide for the Care and Use of Laboratory Animals, and with the guidelines for the care and use of animals established by the Animal Care and Use Committee of Gifu University (Gifu, Japan).

2.2. Reagents

Alpha-galactosylceramide (GalCer) was obtained from Funa-koshi Co. Ltd. (Tokyo, Japan) and stored as a 200

m

g/ml stock so-lution in dimethyl sulfoxide (DMSO). Isoliquiritigenin (ISL), a NALP3 inhibitor, was purchased from Sigma-Aldrich (St Louis, MO). 2.3. Animal treatment

GalCer was diluted in pyrogen-free saline to obtain the indicated dose just before the intraperitoneal injection of a total volume of 200

m

l/mouse. Hepatocellular injury was monitored biochemically through measurement serum alanine aminotransferase (ALT) ac-tivity. At appropriate time points, mice were killed by cervical dislocation and necropsy was performed. Liver tissue samples were ﬁxed in 10% neutral buffered formalin, embedded in parafﬁn, and sectioned; the sections were then stained with hematoxylin and eosin.

2.4. Measurement of serum transaminase

Hepatocyte damage was assessed at the indicated time points after GalCer injection through measurement of serum ALT activity using an automated clinical analyzer (BM2250; JEOL Ltd., Tokyo). 2.5. Real-time reverse transcription (RT)ePCR

Total RNA was extracted from mouse liver using ISOGEN II re-agent (Nippon Gene, Tokyo, Japan), and reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The resulting cDNA was used as a template for real-time RT-PCR with primer-robe sets for IL-1

b

, TNF-

a

, CCL2, MIP1a, CXCL10, IFN-

g

, and 18S rRNA, the latter as an internal control, according to the manufacturer's instructions (TaqMan®Gene Expression Assays and Universal PCR Master mix; Applied Biosystems). Real-time RT-PCR was carried out using the Light-Cycler® 480 system (Roche Diagnostic Systems, Basel, Switzerland).

2.6. Enzyme-linked immunosorbent assay (ELISA)

IFN-

g

concentration in culture supernatant and serum from GalCer treated mice were measured using ELISA. Ninety-six-well

Immulon 2HB plates (Thermo Lab Systems, OH, USA) were coated with puriﬁed IFN-

g

-speciﬁc antibody and then blocked with phosphate-buffered saline (PBS) containing 1% bovine serum al-bumin (BSA). Culture supernatants or mouse serum were then added to the wells of the ELISA plate, and bound cytokine was detected by the addition of biotin-labeled secondary IFN-

g

-speciﬁc antibody (all antibodies were obtained from eBioscience), followed by phosphatase-conjugated avidin and a phosphatase-speciﬁc colorimetric substrate (Sigma-Aldrich). Standard curves were generated using recombinant cytokines purchased from R&D Systems.

2.7. Western blot analysis

Protein (20

m

g) from lysed wound sites was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After blocking non-speciﬁc reaction with 5% skim milk, the membrane was incubated with anti-pro-caspase-1, anti-cleaved caspase1(p20), precursor

IL-Fig. 1. Activation of inﬂammasomes in the liver of WT and NALP3-KO mice after GalCer injection (A) Expression of pro-caspase 1 and p20 protein in the livers of WT and NALP3-KO mice 24 h after treatment with GalCer was examined by western blot analysis and was determined using the GAPDH protein. Data are representative of at least 3 independent experiments with similar results. (B) Expression of precursor IL-1b in the livers of WT and NALP3-KO mice 24 h after treatment with GalCer was examined by western blot analysis and was determined using the GAPDH protein. Data represent at least 3 independent experiments. Each value is represented by the mean (SD) of three mice. *, p< 0.05.

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1

b

. And anti-GAPDH antibodies for 60 min at room temperature and subsequently incubated with peroxidase-labeled anti-mouse or -rabbit IgG antibody for 60 min at room temperature. Immu-noreactive protein bands were visualized with ECL plus (GE Healthcare UK Ltd., England).

2.8. Hepatic mononuclear cell preparation andﬂow cytometric analysis

Hepatic mononuclear cells (MNCs) were isolated and puriﬁed as previously described [13]. Brie_{ﬂy, the excised liver was cut into} small pieces with scissors, pressed through a 200-gauge stainless steel mesh, and suspended in PBS. Lymphocytes were separated from parenchymal hepatocytes and hepatocyte nuclei by Ficoll-Conray (IBL, Gunma, Japan), and washed twice in ice-cold

medium. Cell viability and cell number were assessed by trypan blue exclusion. Forﬂow cytometry, 2 105_{liver MNCs were stained}

using a standard protocol. The following antibodies were used: ﬂuorescein isothiocyanate (FITC)-labeled rat anti-mouse Gr-1 mAb (clone RB6-8C5; Biosciences) and, phycoerythrin (PE)-labeled rat anti-mouse CD11b mAb (clone M1/70; Biosciences). Samples were measured on a FACStarﬂow cytometer and data analysis was per-formed using CellQuest software (BD Biosciences).

2.9. Statistical analysis

Values are expressed as the mean± SD. Differences between experimental and control groups were analyzed by the Kruskal-Wallis test followed by Scheffe's test. A value of p < 0.05 was considered statistically signiﬁcant.

Fig. 2. Induction of liver injury by GalCer in WT mice and IDO-KO mice. (A) Serum ALT activity was measured 0, 1, 2, and 7 days after GalCer injection into WT and NALP3-KO mice. Each value is represented by the mean (SEM) of nine mice. *, p< 0.05. (B) Histopathological characteristics of WT and NALP3-KO mice livers observed at 0, 1, and 2 days after GalCer administration. (hematoxylin and eosin, scale bars in the panel: 100mm). These experiments were repeated 3 times, and the same results were obtained. (C) The number of intrahepatic mononuclear cells (MNCs) in WT and NALP3-KO mice after GalCer injection. (D) Serum ALT activity was measured 0, and 1 days after GalCer injection into WT mice and isoliquiritigenin (ISL)-treated WT mice. Each value is represented by the mean (SD) of three mice. *, p< 0.05.

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3. Results

3.1. Activation of the NALP3 inﬂammasome in the liver after systemic administration of GalCer

Wefirst measured the expression of pro-caspase-1 and cleaved pro-caspase-1 (p20) by western blot to examine inflammasome activation in the liver after the administration of GalCer. As shown inFig. 1A, the expression of p20 in WT mice significantly increased after GalCer injection. The activation of caspase-1 cleaves pro- IL-1

b

into its mature form. Which is then is rapidly secreted from the cells [14]. Thus, we examined the expression of pro-IL-1

b

signiﬁcantly increased in GalCer treated NALP3-KO mice (Fig. 1B).

3.2. GalCer-induced liver injury in WT mice and NALP3-KO mice To clarify the role of inflammasome activation in Galcer-induced liver injury, serum ALT levels were measured after GalCer admin-istration to both WT and NALP3-KO mice. As shown inFig. 2A and days after GalCer injection, ALT levels signi_{ficantly decreased in} NALP3-KO mice. Consistent with thisfinding, histological exami-nation revealed that the number of necrotic foci in NALP3-KO mice were significantly reduced 1 day after GalCer injection (Fig. 2B). The number of inflammatory foci after GalCer injection also decreased in NALP3-KO mice. Necroinflammatory foci are defined as the infiltration of inflammatory cells into a region in which more than three, hepatocytes are undergoing necrosis, and inflammatory cell foci are defined as the existence of more than ten inflammatory cell in one paraffin sections of the liver. The data are represented as the numerical value per area (1 mm2). Additionally, we counted the number of intrahepatic MNCs in WT and NALP3-KO mice after isolating MNCs. The number of MNCs also decreased in NALP3-KO mice after GalCer injection (Fig. 2C). Next, we used ISL, which is a potent inhibitor of NALP3 signaling, to confirm data from the

examination of NALP3-KO mice. Pre-treatment using ISL markedly reduced serum ALT levels in WT mice in response to GalCer in-jection (Fig. 2D).

3.3. Cytokine and chemokine expression in WT mice and NALP3-KO mice treated with GalCer

As previously reported, GalCer administration induced various proinﬂammatory cytokines and chemokines in the liver [4,13]. Therefore, we measured mRNA expression of TNF-

a

, IL-1

b

, CCL2, MIP1

a

, and CXCL10 in the liver of GalCer-treated WT and NALP3-KO mice. Intrahepatic expression of TNF-

a

mRNA and CCL2 was signiﬁcantly decreased in NALP3-KO mice compared with that WT mice 1 day after the administration of GalCer.

3.4. Increased frequency of CD11bþ/Gr-1intermediatecells in NALP3-KO mice compared with WT mice

A recent report found that the frequency of CD11bþ/Gr-1

inter-mediate_{cells decreased in NALP3-KO mice compared with WT mice}

[15]. In addition, CD11bþ/Gr-1intermediatecells have been shown to suppress the immune response via the reduction of IFN-

g

pro-duction [16]. Therefore, we next examined the frequency of CD11bþ/Gr-1intermediatecells in hepatic MNCs from WT and NALP3-KO mice treated with GalCer. As shown inFig. 4A, the frequency of CD11bþ/Gr-1intermediatecells in NALP3-KO mice was much higher than in WT mice, both before and after GalCer injection. Both mRNA and protein level expression of IFN-

g

in the liver of GalCer-treated NALP3-KO mice were signiﬁcantly lower than in WT mice. More-over, GalCer-induced IFN-

g

production in splenocytes in vitro was signi_{ﬁcantly lower in NALP3-KO mice was compared with WT mice} (Fig. 4B, C, and D).

Fig. 3. The relative mRNA expression of TNF-a, IL-1b, CCL2, MIP1a, and CXCL10 in the liver of GalCer-treated WT and NALP3-KO mice were measured using quantitative real-time RT-PCR. The results were normalized to the expression of 18S rRNA. Each value is represented by the mean (SD) ofﬁve mice. *, p < 0.05.

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4. Discussion

In this study, we demonstrated that either genetic or chemical inhibition of NALP3 activity can reduce the inflammatory response and liver injury. This NALP3 inhibition impaired GalCer induced recruitment of inflammatory cells into the liver by reducing in-flammatory chemokine expression. Interestingly, the number of CD11bþ/Gr-1intermediatecells, which have an immune-suppressive effect, increased in GalCer-treated NALP3-KO mice, and these cells may be involved in the prevention of more severe GalCer-induced liver injury in NALP3-KO mice. To the best of our knowledge, this is thefirst report describing the role of NALP3 in the development of GalCer-induced liver injury.

Induction of in_{ﬂammasomes complexes is required to produce}

mature IL-1

b

and IL-18. Which are involved in many immune-mediated diseases. For example, IL-1

b

is a marker of RA progres-sion RA, and RA patients also show signs of inﬂammasome acti-vation[17,18]. In the present study, the activated form of caspase-1 (p20) increased after GalCer injection (Fig. 1). Moreover, the expression of pro-IL-1

b

in GalCer-treated NALP3-KO mice increased compared with GalCer-treated WT mice. Real-time RT-PCR analysis revealed that IL-1

b

mRNA expression increased in both WT and NALP3-KO mice after GalCer injection (Fig. 3). Accordingly, pro-IL-1

b

protein levels also increased in GalCer-treated WT and NALP3-KO mice compared with non-treated mice. These results indi-cated that GalCer administration promotes inﬂammasome activa-tion in WT mice, and pro-IL-1

b

accumulated in GalCer-treated NALP3-KO mice due to the absence of the in_ﬂammasome.

Fig. 4. The frequency of CD11bþ/Gr-1intermediate_{cells in WT mice and NALP3-KO mice treated with GalCer. (A) The frequency of CD11b}þ_/Gr-1intermediate_{cells in intrahepatic MNCs of}

WT and NALP3-KO mice after GalCer injection was examined usingﬂow cytometry. (B) The relative expression levels of IFN-gmRNAs in the liver of WT and NALP3-KO mice after GalCer injection were measured using quantitative real-time RT-PCR. The results were normalized to the expression of GAPDH. (C) The IFN-glevels in the serum from WT and NALP3-KO mice after GalCer injection were determined using ELISA. (D) Splenocytes (1 10gcells/ml) from WT and NALP3-KO mice were cultured with or without GalCer (100 ng/ ml) for 24 h. The concentration of IFN-gin culture supernatant was determined using ELISA. The data are presented as the means± SD of the results of three samples from each group. *p< 0.05.

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Inﬂammasome activation is involved in the production of IL-1

b

and IL-18. Previous studies have demonstrated that expression of IL-1

b

expression is increased in the liver and the serum of patients with hepatitis[19,20], and an IL-18 neutralizing antibody attenu-ated liver injury in a Con A-induced hepatitis model[21]. IL-1

b

and IL-18 are also involved in the progression of other immune-mediated diseases. An IL-1 receptor antagonist has been shown to be effective in patients with RA[22,23]. Thus, IL-1

b

and IL-18 play a critical role in immune-mediated diseases, and inflammasomes that regulate the production and maturation of these cytokines are also involved in the progression of such diseases. In the present study, GalCer-induced liver injury was significantly attenuated in NALP3-KO mice compared with WT mice (Fig. 2). Histological ex-amination and real-time RT-PCR analysis indicated that NALP3 deficiency reduced liver inflammation after GalCer injection. Therefore, the reduction of inflammation may lead to the attenu-ation of GalCer-induced liver injury.

Histological examination, found fewer immune cells inﬁltrating the liver in treated NALP3-KO mice compared with GalCer-treated WT mice. This reduction was partially related to the decrease in chemokine expression in NALP3-KO mice injected with GalCer (Fig. 3). The absolute number of intrahepatic MNCs was also reduced in NALP3-KO mice compared with WT mice. Interestingly, the frequency of CD11bþ/Gr-1intermediatecells after GalCer treatment increased in NALP3-KO mice compared with WT mice. Consistent with these results, Chow MT et al.[24]demonstrated that the fre-quency of CD11bþ/Gr-1intermediate_{cells increased in tumor-bearing}

NLRP3-KO mice compared with WT mice. Myeloid-derived sup-pressor cells (MDSCs) are immunosuppressive and have been shown to express CD11b and Gr-1 on their cell surfaces. CD11bþ /Gr-1þcells are divided into two populations: CD11bþ/Gr-1highcells and CD11bþ/Gr-1intermediatecells. A recent report demonstrated that the number of CD11bþ/Gr-1intermediatecells increased in lethal tuber-culosis infection in mice[16]. In mice infected with Mycobacterium tuberculosis these CD11bþ/Gr-1intermediatecells suppressed IFN-

g

production from bone marrow cells and the subsequent activation of immune cells. Thus, the accumulation of CD11bþ/Gr-1intermediate cells in the liver may suppress the inﬂammatory response by reducing IFN-

g

. Therefore, the accumulation of CD11bþ/Gr-1

inter-mediate_{cells in NALP3-KO mice is not a little involved in the}

atten-uation of GalCer-induced liver injury in NALP3-KO mice. By contrast, another study demonstrated that the activation of in_{ﬂammasomes impaired immune suppression in MDSCs in GVHD} mouse model[25]. Thus, it remains unclear how the inhibition of NALP3 signaling affects MDSC development, and further studies are needed.

Although there is no essential therapy for AIH, corticosteroid treatment is often used and effective for the control of AIH[26]. However, corticosteroid treatment has many side effects, and several support therapies are required for these side effects. Thus, new treatment strategies for AIH must be developed, and this re-quires identifying and inhibiting the molecules involved in AIH progression.

In the present study, we revealed the role of NALP3 in GalCer-induced liver injury. The inhibitor of NALP3 signiﬁcantly reduced liver injury after the administration of GalCer (Fig. 2D). And decreased the expression of pro-inﬂammatory cytokines and che-mokines, leading to the recruitment suppression of immune cells into the liver. Moreover, the frequency of CD11bþ/Gr-1intermediate MDSCs increased in GalCer-treated NALP3-KO mice. The increase of MDSCs may be involved in the attenuation of GalCer-induced liver injury in the absence of NALP3 activation. These observations indicate that inhibition of NALP3 signaling could be a therapeutic target in AIH.

Conﬂict of interest

All the authors declares no conﬂict of interest. Acknowledgements

We thank Genentech, Inc. (South San Francisco, CA) for providing the NALP3-KO mice.

Transparency document

Transparency document related to this article can be found online athttp://dx.doi.org/10.1016/j.bbrc.2017.06.049.

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