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Supporting Information Supporting Information Sarashina-Kida et al. 10.1073/pnas.1712837114 Reagents and Cells merged using the fastq-join program based on overlapping se- Dexametazone was purchased from Sigma. Human recombinant quences. Reads with an average quality value of <25 and inexact FGF7, FGF10, and EGF were purchased from R&D. OCUG1 matches to both universal primer were filtered off. Filter-passed was obtained from the Japanese Collection of Research Bio- reads were used for further analysis after trimming off both resources cell bank. Huh7 was described previously (29). Metyr- primer sequences. For each sample, 10,000 quality filter-passed apone was purchased from Cayman. The 11β-hydroxysteroid reads were rearranged in descending order according to the dehydrogenase type 1 (11β-HSD1) inhibitor PF-915275 was pur- quality value and then clustered into operational taxonomic chased from Santa Cruz. units (OTUs) with a 96% pairwise-identity cutoff using the UCLUST program (Edgar 2010) version 5.2.32 (www.drive5. RNA Analysis com/). Taxonomic assignments of each OTU were made by RNA was extracted by using RNeasy (Qiagen) and subjected to similarity searching against the Ribosomal Database Project reverse transcription (RT) with PrimeScript RT Master Mix (TaKaRa (RDP) and the National Center for Biotechnology Information Bio) according to the manufacturer’s protocol. Quantitative RT-PCR genome database using the GLSEARCH program. The 16S (qRT-PCR) was performed with a LightCycle480 instrument rRNA gene V1–V2 region sequences analyzed in this study and SYBR Green reagents (Roche Molecular Biochemicals). were deposited in DDBJ/GenBank/EMBL with the accession Data are presented as relative expression units after normal- nos. BioProjectID PRJDB5950. ization to Gapdh. Primer sequences for Il6, Tnf,andGapdh have been described previously (4). Primer sequences for the Bacterial Proliferation Assay Sftpd gene are as follows: Sftpd forward 5′-CCAGTTGGACC- Frozen stock of L. murinus or L. johnsonii was suspended in CAAAGGAGAGAATG-3′ and Sftpd reverse 5′-GTCCTATG- 10 times volume of Lactobacilli MRS broth (BD Biosciences) TTCCCCTGCTTCCCA-3′. with 2.7 mM MgCl2 and incubated in the presence of indicated concentration of recombinant SP-D (R&D) at 37 °C for in- Histological Analysis dicated time periods. Relative viability of bacteria was examined Isolated gallbladders were fixed in 4% paraformaldehyde for 16 h by CTC assay kit (Dojindo Molecular Technologies) according at room temperature, embedded in paraffin, and stained with to the manufacturer’s protocol. OD595 was measured by hematoxylin and eosin (H&E). Immunohistochemical analysis microplate reader (Bio-Rad Laboratories). To examine in- of SP-D was performed with mouse monoclonal anti–SP-D Ab terference of the growth of Clostridia species by L. murinus,100μL (12G5; Abcam) or isotype mouse IgG1 (3A1; Cell Signaling) and of confluent culture of Clostridia was diluted by 10 times with HRP-conjugated secondary Ab to visualize signal using DAB Eggerth-Gagnon (EG) broth and incubated in the absence or substrate. The H&E or immunohistochemical sections were ex- presence of confluent culture of L. murinus with indicated vol- amined by microscope (Nikon ECLIPSE Ti). ume at 37 °C under anaerobic conditions (23). After 24 h, bac- terial DNA was extracted by phenol/chloroform method and ELISA for SP-D relative DNA amounts of Clostridial species were examined by Concentration of biliary SP-D was measured by SP-D ELISA kit qPCR with the following primers: C. bolteae forward 5′-CG- (Yamasa) according to the manufacturer’s protocol. GCGTGCCTAACACAT-3′ and C. bolteae reverse 5′-GTCC- GCCACTCAGTCAATCA-3′; C. hathewayi forward 5′-CGA- Fungal DNA Analysis GCGAAGCGGTTTCA-3′ and C. hathewayi reverse 5′-TTC- Fungal DNA was extracted by QIAamp DNA Stool Mini Kit TAACTGTTATCCCCCAGTGTA-3′;andC. 7 3 54FAA forward (Qiagen). Primer sequences for Candida tropicalis, Saccharomycopsis 5′-AGTCGAACGAAGCGATTTAAC-3′ and C. 7 3 54FAA re- fibuligera,andSaccharomyces cerevisiae have been described pre- verse 5′-CCGGAGTTTTTCACACTGTAT-3′. viously (9, 30). Primer sequences for other microbes are as follows: Wickerhamomyces anomalus forward 5′-GCGATAAACCTTACA- Isolation of Lamina Propria Cells CACATTGTCTA-3′ and Wickerhamomyces anomalus reverse 5′- The detailed protocol was previously described (32). The pro- CAGTAAGCCAGGCTCACCA-3′; Debaryomyces hansenii forward cedure is briefly described as follows: After fecal content was re- 5′-CTTGGTTGGGTTCCTCGCA-3′ and Debaryomyces hansenii moved, whole colonic tissue was incubated in EDTA-containing reverse 5′-CGAAGTAGAGCCACATTCCTTAGT-3′; Trichospor- buffer to remove intestinal epithelial cells. The remaining on moniliiforme forward 5′-TTCTTAATGGCTTGGAATTGGG- lamina propria with muscles was cut into small pieces and TGT-3′ and Trichosporon moniliiforme reverse 5′-TTAGAAG- digested with collagenase D, DNaseI, and dispase. After being CGTACTTCTCAAGCCGAC-3′; Trichosporon domesticum forward washed, cell suspension was subjected to gradient separation to 5′-AGGATCATTAGTGATTGCCTTAATTG-3′ and Trichosporon obtain lamina propria cells in the layer between upper and domesticum reverse 5′-ACAATGTTTGTATAAAATCGAATCCG-3′; lower phases. and Trichosporonales forward 5′-GCGATAAGTAATGTGAA- TTGCAGA-3′ and Trichosporonales reverse 5′-AAGAAACCC- Scanning Electron Microscopy TAATGGTTGAGATTT-3′. The 1 × 108 cfu of L. murinus and Clostridia species were in- cubated with BSA (5 μg/mL) or SP-D (5 μg/mL) for 1 h in PBS Microbiota Analysis (0.9 mM CaCl2). Samples were placed on a plastic coverslip Bacterial DNA was extracted from the feces using the enzymatic (Celltight C-1 Cell Desk LF; Sumitomo Bakelite Co.) and lysis method described previously (31). Hypervariable regions centrifuged at 700 × g for 5 min at 4 °C. After several wash steps, (V1–2) of 16S rRNA gene were PCR amplified using primers samples were fixed with 2% formaldehyde and 2.5% glutaralde- 27Fmod 5′-agrgtttgatymtggctcag-3′ and 338R 5′-tgctgcctcccgt- hyde in 0.1 M phosphate buffer (pH 7.4), postfixed with 1% os- aggagt-3′. The 16S metagenomic sequencing was performed mium tetroxide and 0.5% potassium ferrocyanide in the same according to the Illumina protocol. Two paired-end reads were buffer, treated with 1% tannic acid, and retreated with 1% Sarashina-Kida et al. www.pnas.org/cgi/content/short/1712837114 1of7 osmium tetroxide. Samples were dehydrated in a graded series with a S-4800 field emission scanning electron microscope (Hitachi of ethanol and substituted with liquefied carbon dioxide in a High-Technologies Corp.). chamber device, Samdri-PVT-3D (Tousimis Research Corp.). After critical point drying, samples on the coverslip were mounted Statistical Analysis on an aluminum specimen block with carbon adhesive tape and Differences between control and experimental groups were evaporated osmium tetroxide vapor. Sample cells were observed evaluated by Student’s t test. Sarashina-Kida et al. www.pnas.org/cgi/content/short/1712837114 2of7 (A) (B) 0.50 *** p=0.068 5.0 * 4.0 0.45 3.0 0.40 2.0 0.35 Shannon index 1.0 Weighted unifrac distance 0.30 0 KO KO WT WT Sftpd -/- KO WTWT (C) List of bacteria that significantly changed in Sftpd-/- mice -/- WT Sftpd OTU number Closest related strains Phylum Order total read total read p -value counts counts Increased in Sfptd -/- mice OTU00007 Lactobacillus murinus (100%) Firmicutes Lactobacillales 133 12815 0.0141 OTU01291 Lactobacillus animalis (96.99%) Firmicutes Lactobacillales 10 2562 0.0129 OTU00840 Lactobacillus murinus (96.99%) Firmicutes Lactobacillales 4 2528 0.0131 OTU00201 Prevotella sp. oral taxon 317 (79.94%) Bacteroidetes Bacteroidales 337 1055 0.0070 OTU00402 Prevotella sp. P2A_FAAD4 (84.91%) Bacteroidetes Bacteroidales 0 1160 0.0414 OTU02031 Bacteroides acidifaciens (83.86%) Bacteroidetes Bacteroidales 145 509 0.0136 OTU00288 Alistipes senegalensis (90.57%) Bacteroidetes Bacteroidales 58 416 0.0027 OTU00155 Clostridium bolteae (88.82%) Firmicutes Clostridiales 0 340 0.0027 OTU00728 Barnesiella viscericola (80.31%) Bacteroidetes Bacteroidales 70 223 0.0405 OTU00121 Rikenella microfusus (86.35) Bacteroidetes Bacteroidales 0 179 0.0015 OTU01169 Lactobacillus johnsonii (96.46%) Firmicutes Lactobacillales 6 168 0.0003 Decreased in Sfptd -/- mice OTU00128 Clostridium bolteae (87.96%) Firmicutes Clostridiales 3152 46 0.0106 OTU00285 Prevotella oris (79.94%) Bacteroidetes Bacteroidales 1273 0 0.0309 OTU00529 Clostridium hathewayi (85.37%) Firmicutes Clostridiales 1071 14 0.0383 OTU00183 Bacteroides acidifaciens (99.37%) Bacteroidetes Bacteroidales 999 347 0.0412 OTU01895 Clostridium bolteae (87.65%) Firmicutes Clostridiales 462 6 0.0110 OTU01148 Barnesiella intestinihominis (83.18%) Bacteroidetes Bacteroidales 412 60 0.0397 OTU00181 Prevotella shahii (86.12%) Bacteroidetes Bacteroidales 386 114 0.0455 OTU00151 Prevotella genomosp. C1 (85.89%) Bacteroidetes Bacteroidales 298 72 0.0327 OTU00194 Eubacterium coprostanoligenes (84.4%) Firmicutes Clostridiales 232 0 0.0414 OTU00011 Clostridium sp. ASF356 (95.87) Firmicutes Clostridiales 175 20 0.0401 * OTUs that display a significant increase or decrease in read counts in Sfptd-/- mice compared to wild type (WT) mice in 16s ribosomal RNA sequence analysis shown in Fig. 1E. Total read counts of each OTU listed are more than 0.1% of those of all OTUs. Fig. S1. The 16s ribosomal RNA (rRNA) sequence analysis shown in Fig. 1E. Weighted uniFrac (A) and Shannon index (B) are shown. ***P < 0.001, *P < 0.05. (C) − − OTUs that display a significant increase or decrease in read
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