Mice. Clec7a–/– mice have been described (40). Heterozygous (Clec7a+/–) mice on a C57BL/6 genetic background were bred to generate mice without disruption of Clec7a (WT) and mice with homozygous disruption (Clec7a–/– mice). Female mice (age, 8 weeks) were fed ethanol for 8 weeks (the Lieber-DeCarli diet model of chronic alcohol consumption), as previously described (23). The Lieber-DeCarli diet comprises Micro-Stabilized Alcohol Rodent Liquid Diet (LD101A, irradiated; TestDiet), maltodextrin IRR (9598; TestDiet), and 200-proof ethanol (Gold Shield). The caloric intake from ethanol was 0 on day 1, 10% of total calories on days 2–4, 20% on days 5–7, 30% from day 8 until the end of 6 weeks, and 36% for the last 2 weeks. Control mice received an isocaloric amount of isomaltose instead of ethanol.

To produce bone marrow chimeras, mice were given lethal doses of radiation (600 rad) twice, using a 137Cs source. Two weeks after bone marrow transplantation, mice were injected i.p. with 200 μl of clodronate liposomes (5 mg/ml; Vrije Universiteit, Amsterdam, Netherlands) to deplete radioresistant Kupffer cells. The Lieber-DeCarli diet began 4 weeks after bone marrow transplantation.

To reduce intestinal commensal fungi, nonabsorbable amphotericin B (60 mg/kg/d) (41) (Medisca) was added to the liquid Lieber-DeCarli alcohol-containing and control diets. Female C57BL/6 mice were purchased from Charles River Laboratories for this experiment.

To prove whether soluble β-glucan can activate CLEC7A in vivo, carboxymethyl-β-1,3-d-glucan (2 mg; CM-curdlan, Wako) was injected i.p. into WT and Clec7a–/– mice. Control animals received a comparable vehicle (0.01 M NaOH, saline; pH 7) treatment. Mice were sacrificed 2, 8, and 24 hours after CM-curdlan injection and investigated for plasma IL-1β levels and hepatic Il1b expression.

Real-time qPCR. RNA of mouse tissues was extracted using Trizol (Invitrogen). DNase treatment of RNA was performed using the DNA-free Kit (Ambion), and RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (ABI). Real-time qPCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad) using primer sequences obtained from NIH qPrimerDepot (mouse and human) and a StepOnePlus thermocycler (ABI). The qPCR value was normalized to mouse 18S. Gene expression results are expressed relative to the levels of control (WT) mice or cells. DNA was isolated from feces as previously described (4, 21, 42). Published microbial primer sequences for 16S rRNA gene (43) and fungal-specific 18S rDNA gene were used (44). To quantify the total bacterial or fungal load present in feces, the qPCR value of fungal 18S rRNA gene or the bacterial 16S rRNA gene, respectively, was multiplied by the total amount of DNA (micrograms) per milligram of feces for each sample.

Sequencing and analysis. We performed deep DNA pyrosequencing of fecal DNA, targeting the hypervariable V4 region of prokaryotic 16S rRNA loci, using a dual indexing strategy on the Illumina MiSeq platform, as previously described (45). Microbial community profiles were generated using species-level (97% similarity) operational taxonomic unit–based (OTU-based) classification and analysis, as previously described by us (22). Sequence data were registered at NCBI under BioProject PRJNA317649. Sequence reads are available at NCBI under the following consecutive BioSample IDs: SAMN04874715–SAMN04874755.

To evaluate the human intestinal mycobiome, fungal-specific internal transcribed spacer (ITS) amplicon sequencing and analysis were conducted as previously described (4, 46–48). In brief, ITS sequence data were generated with Illumina MiSeq V2 kit using primers BITS and B58S3 specific for ITS1 (47), by a sequencing dual indexing strategy similar to that used for 16S rRNA. Specifically, the 3 primers used are as follows (pad in regular type, linker in brackets, and targeting primer sequence in boldface): read 1 (ITSFWD, containing BITS), CATTATAGCT[CA]ACCTGCGGARGGATCA; I7 index, AACTTTYARCAAYGGATCTC[TG]CTGTATGACT; and read 2 (ITSREV, containing B58S3), AGTCATACAG[CA]GAGATCCRTTGYTRAAAGTT. Index 2 was the standard Illumina Index 2 sequencing primer. PCR parameters included a 95°C denaturing step followed by 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension step of 72°C for 5 minutes. Amplicons were purified using the GeneJet PCR purification system (Thermo Fisher Scientific). Purified amplicons were then quantified using SYBR Gold (Thermo Fisher Scientific), normalized, and pooled to generate a library of equimolar DNA molecules per sample.

To evaluate the mouse intestinal mycobiome, which has a lower abundance of fungi than that of humans, we optimized DNA extraction, quantitation, and amplification methods. Samples were vortexed in Qbiogene lysing matrix B tubes using a Mini-Beadbeater instrument (Biospec Products). We then followed a published DNA isolation protocol (8). DNA samples were quantified using PicoGreen dsDNA assay (Life Technologies); 100 ng DNA was used for PCR with Q5 High Fidelity Polymerase (NEB) with BITS and B58S3 primers specific for ITS1 containing partial dual-indexed Illumina adapters as for 16S rRNA (47) under the following conditions: 98°C denaturing step followed by 39 cycles of 98°C for 10 seconds, 55°C for 20 seconds, and 72°C for 30 seconds, followed by a final extension step of 72°C for 2 minutes. Amplicons were purified twice with Agencourt AMPure beads (Beckman Coulter) at 0.9 beads to DNA ratio. To assess whether amplicons were free of adapter dimers and to verify insert size, amplicons were analyzed and quantified on the Bioanalyzer, using the high-sensitivity dsDNA chip (Agilent), and pooled. The final pool was quantified with Library Quant kit (Kapa Biosystems). Sequences were generated using the MiSeq 2*150 V2 kit (Illumina) with 35% PhiX library added and custom primers spiked in. The ITS and index primer sequences were the same as those used for the human mycobiome.

Sequence processing and analysis steps were performed by our open-access, mothur-based (49) workflow software, YAP (46, 50). Mothur version 1.34 was used in this study. To achieve acceptable memory and run-time scalability with large MiSeq data sets, the preclustering and clustering stages of the pipeline were implemented with CD-HIT (51, 52) sequence clustering software instead of the corresponding built-in mothur commands. Taxonomic classification of assembled forward and reverse reads was performed by mothur’s implementation of the RDP Naïve Bayesian Classifier (53) using version 1.2 of the manually curated targeted host-associated fungi (THF) database (54), trimmed to include only the ITS1 region amplified by the BITS/B58S3 primers, removal of non-ITS sequences, and trimmed sequences less than 45 nucleotides in length, and then formatted for use with mothur. Fungal species-level OTUs were defined at 97% sequence identity (47). Candida species-level classification of ITS sequences was performed using STIRRUPS (55) with a custom sequence library of 31 nonredundant representative sequences pulled from the trimmed THF database. Sequence data were registered at NCBI under BioProject PRJNA317653. Sequence reads are available from the NCBI under the following consecutive BioSample IDs: SAMN04874830–SAMN04874861.

Protein analysis. IL-1β (14-7012, clone B122; eBioscience) and albumin (E90-134; Bethyl Labs) were measured by ELISA. Human serum samples were assessed for the presence of ASCA-IgG using ELISA (KA1270; Abnova). Immunoblot analysis was performed using antibodies against cytochrome P450 family 2 subfamily E polypeptide 1 (AB1252; CYP2E1; Millipore), β-actin (A5441; Sigma), occludin (71-1500; Invitrogen), NLRP3 (MAB7578; R&D Systems), and caspase-1 p20 (AG-20B-0042-C100; AdipoGen). For detection of cleaved caspase-1 in culture supernatant, proteins were precipitated using methanol/chloroform, and whole extracts were subjected to immunoblotting. Immunoblots were analyzed by densitometry, using NIH ImageJ.

Biochemical analysis. Plasma levels of alanine aminotransferase (ALT) and hepatic levels of triglyceride were measured using the Infinity ALT kit (Thermo Fisher Scientific) and Triglyceride Liquid Reagents Kit (Pointe Scientific), respectively. Plasma levels of ethanol were measured using the Ethanol Assay Kit (BioVision). Hepatic alcohol dehydrogenase (ADH) activity was determined using the ADH Assay Kit (BioVision). Plasma LPS (CEB526Ge; Cloud-Clone Corp.) and 1,3-β-d-glucan (MBS730464; MyBioSource) levels were measured by ELISA. Plasma was diluted 1:4 for 1,3-β-d-glucan measurements.

Staining procedures. For immunofluorescence staining, antigens of formalin-fixed slides were retrieved with a retrieval solution (Dako) containing 1% Triton and blocked using tissue blocking solution (Dako). F4/80 antibody (14-4801; eBioscience) and antibody against IL-1β (ab9722; Abcam) were used as primary antibodies. Nuclei were stained with DAPI (blue). For histologic analysis of liver tissues, formalin-fixed liver sections were stained with H&E. Frozen liver sections were analyzed by Oil Red O staining. Samples were analyzed by densitometry, using NIH ImageJ. The results are presented as percentage area positively stained. Immunofluorescent staining of mouse and human liver was performed using frozen sections with primary antibodies against CLEC7A (MCA2289FT; conjugated with FITC; Bio-Rad), mouse F4/80 (14-4801; eBioscience), and human CD68 (IS613; Dako).

Mouse cell culture studies. Primary mouse Kupffer cells, hepatic stellate cells, and hepatocytes were isolated as previously described (56) with minor modifications. RNA was extracted from hepatic stellate cells directly after the isolation. Quiescent hepatic stellate cells were isolated from C57BL/6 mice, and activated hepatic stellate cells were purified from mice exposed to carbon tetrachloride. C57BL/6 mice were gavaged with 200 μl carbon tetrachloride (25% vol/vol in corn oil) twice weekly for 6 weeks (57). Kupffer cells were isolated from C57BL/6, Clec7a–/–, or Casp1–/– mice (which are also Casp11-deficient) (generated by R. Flavell, Yale University, New Haven, CT, USA) (58) and selected by magnetic cell sorting using anti-CD11b Micro Beads (Miltenyi Biotec). Kupffer cells were cultured with RPMI 1640 (Thermo Fisher Scientific) containing 10% FBS for 4 hours. After an overnight starvation in medium without FBS, Kupffer cells were stimulated with curdlan (100 μg/ml; InvivoGen) for 4 hours (gene expression) or 8–9 hours (protein secretion experiments, immunoblotting experiments).

Primary mouse hepatocytes were cultured with Medium 199 (Thermo Fisher Scientific) containing 10% FBS for 4 hours. After overnight starvation in medium without FBS, cells were stimulated with curdlan (100 μg/ml) for 8 hours, or IL-1β (10 ng/ml; R&D Systems) for 24 hours. Hepatocyte death was quantified using the Pierce LDH Cytotoxicity Detection Kit (Thermo Fisher Scientific). Oil Red O staining was used to identify lipid accumulation in cultured hepatocytes.

For conditioned medium and IL-1β neutralization experiments, Kupffer cells were cultured with RPMI 1640 medium containing 10% FBS for 4 hours and serum starved in RPMI 1640 and Medium 199 (50% vol/vol) overnight. Kupffer cells were then stimulated with curdlan for 8 hours. Cell supernatants were transferred to hepatocytes, and incubated for 24 hours. A neutralizing antibody against IL-1β (10 ng/ml; ab9722; Abcam) or isotype IgG (10 ng/ml, control) was added to the conditioned medium after the transfer. Hepatocyte cytotoxicity and intracellular lipid staining experiments were performed.

Isolation of human liver cells. Primary human hepatocytes, Kupffer cells, and hepatic stellate cells were isolated as previously described (59, 60). RNA extraction and quantitative reverse transcriptase PCR were performed as previously described (60) using specific primers for human CLEC7A (NIH human qPrimerDepot) and 18S RNA (forward: 5′-AAACGGCTACCACATCCAAG-3′; reverse: 5′-CCTCCAATGGATCCTCGTTA-3′). 18S was used for normalization. Liver tissues used in this study were provided anonymized by the Biobank under the Administration of the Human Tissue and Cell Research (HTCR) Foundation at the Hospital of the LMU Munich (http://www.klinikum.uni-muenchen.de/Chirurgische-Klinik-und-Poliklinik-Grosshadern/de/0800-gewebebank/index.html). The framework of HTCR Foundation (http://www.htcr.org) (61) includes obtaining written informed consent from all donors after the nature and possible consequences of the donation were explained. This framework has been approved by the ethics commission of the Faculty of Medicine in the University of Munich (no. 025-12) as well as the Bavarian State Medical Association (no. 11142).

Human serum samples. We analyzed data collected from 28 patients with alcoholic liver cirrhosis from July 1, 2010, through October 31, 2010, as previously described (62). In brief, our analysis excluded patients who tested positive for hepatitis C antibody or hepatitis B surface antigen or had any other liver diseases. Liver cirrhosis was detected by ultrasonography (based on presence of cirrhotic liver parenchyma), presence of esophageal or gastric varices (based on upper endoscopy examination), or splenomegaly (based on imaging studies). Continuous alcohol drinking was defined as ingestion of more than 60 g/d of alcohol for more than 10 years in men and more than 20 g/d for more than 10 years in women. We also analyzed data from 2 control cohorts of similar age and sex distribution: 14 healthy individuals and 43 patients with HBV-related liver cirrhosis who did not consume alcohol. Patient characteristics are presented in Supplemental Table 1.

Patients did not take antibiotics or immunosuppressive agents during the 2 months preceding enrollment. Other exclusion criteria were diabetes, inflammatory bowel disease, known liver disease of any other etiology, and clinically significant cardiovascular, pulmonary, or renal comorbidities. The study protocol was approved by the Ethics Committee of En Chu Kong Hospital, and a written informed consent was signed by each participant after the nature and possible consequences of the studies were explained. The percentages of patients with alcoholic liver cirrhosis surviving for a 66-month period were determined from chart review. The cutoff value (8 U/ml) was the median serum level of ASCA-IgG, and the endpoint was liver-related mortality. During the follow-up period (5.5 years), 13 of 28 patients died. The cause of death was liver-related disease in 12 patients, and 1 patient died from metastatic nasopharyngeal carcinoma. One patient was lost to follow-up and excluded from the survival analysis.

Human fecal samples. Healthy individuals without chronic disease (controls, n = 8), alcohol-dependent patients without evidence of progressive liver disease (nonprogressive liver disease, n = 10), patients with alcoholic hepatitis (alcoholic hepatitis, n = 6), and patients with alcoholic liver cirrhosis (alcoholic cirrhosis, n = 4) were included in the study. Diagnosis of alcoholic hepatitis was made by biopsy in 3 of 6 patients. The remaining patients with alcoholic hepatitis and all patients with alcoholic cirrhosis were diagnosed based on a combination of clinical, biochemical, and imaging parameters. All patients included were actively drinking. Baseline features of this cohort are provided in Supplemental Table 2. Stool samples were collected, and DNA was extracted from fecal samples (21). Written informed consent was signed by each participant after the nature and possible consequences of the studies were explained. The study protocol was approved by the IRB of each institution involved.

Human liver and duodenal biopsies. Patients with alcohol dependence and with active alcohol consumption were compared with individuals without alcohol dependency (controls). Duodenal biopsies were taken and immediately snap-frozen from otherwise normal subjects who underwent outpatient upper gastrointestinal endoscopy for gastroesophageal reflux symptoms. Only biopsies from subjects with endoscopically and histologically normal duodenum were finally retained as true controls. Alcohol-dependent patients who followed an inpatient alcohol withdrawal program underwent routine upper gastrointestinal endoscopy with duodenal biopsy on the second day of admission. Patients with disturbed liver function tests and suspicion of significant fibrosis determined noninvasively by Fibroscan were proposed for a liver biopsy. Liver biopsy was performed on the third day of admission in patients. Liver specimens from control patients were obtained from surgical resections or from size-reduced donor livers before transplantation. Written informed consent was signed by each participant after the nature and possible consequences of the studies were explained. The study protocol was approved by the Ethics Committee of the Université Catholique de Louvain.

Statistics. Results are expressed as mean ± SEM. Significance was evaluated using the unpaired Student’s t test except when stated otherwise. For comparison of 3 groups, 1-way ANOVA with Newman-Keuls post-test was used. Not normally distributed (Shapiro-Wilk test) serum levels of ASCA were transformed by taking the logarithm to the base of 10. Significance of mouse microbiome data was determined using the Kruskal-Wallis rank-sum statistical test with YAP. Kaplan-Meier curves were used to compare survival between groups; significance was assessed using the log-rank test. A P value less than 0.05 was considered to be statistically significant.

Study approval. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of UCSD. Studies using human materials were reviewed and approved by an appropriate IRB: (a) ethics commission of the Faculty of Medicine in the University of Munich (no. 025-12) as well as the Bavarian State Medical Association (no. 11142) (human liver cell isolation); (b) Ethics Committee of En Chu Kong Hospital (human serum samples, human cohort study); (c) Human Research Protections Program of UCSD and of the VA San Diego Healthcare System; IRB at Rush University Medical Center; Yale University IRB; University of Wisconsin–Madison Health Sciences IRBs (Madison, Wisconsin, USA); University of North Carolina at Chapel Hill IRB; Ethics Committee of the Université Catholique de Louvain (human fecal samples); (d) Ethics Committee of the Université Catholique de Louvain (human duodenal and liver biopsies). All subjects provided informed consent prior to their participation in the study.