Mice
Dectin-1-deficient mice (D-1KO) were kindly provided by Gordon D. Brown, and Dectin-2-deficient mice (D-2KO) were kindly provided by Mihai G. Netea; these mice were used to establish our colonies of dectin-KO mice, including Dectin-1 and Dectin-2 double-deficient mice (Dectin-1/2 deficient, D-1/2KO) and control littermates animals (wild-type—WT). These mice were generated in a C57BL/6 background and included in the protocol at 8 weeks old. Eight-week-old female wild-type mice were purchased from Janvier Laboratory (Le Genest, France) and used 1 week after reception (WTj). Animals were kept in humidity- and temperature-controlled rooms under a 12-h light-dark cycle and had access to a chow diet and water ad libitum. All experiments were performed with appropriate control groups from the same batch of mice. All experiments were performed in accordance with the ethics committee “Comite d’Ethique en Experimentation Animale” (COMETHEA C2EA – 45, Jouy en Josas, France). Every experiment was repeated at least two times n = 10.
Genotyping of Dectin-1 and Dectin-2 mutants
Mouse ear samples were collected. Ear DNA was extracted according to the recommended method. Briefly, 300 μL of lysis buffer (50 mM Tris-HCl 1 M pH 7.8 (Sigma–Aldrich, St. Louis, MO, USA), 100 mM NaCl (Sigma–Aldrich, St. Louis, MO, USA), 0.5% Tween 20 (Sigma–Aldrich, St. Louis, MO, USA)), and 1.5 μL of proteinase K (Sigma–Aldrich, St. Louis, MO, USA) were added to the tube containing mouse tissue and mixed well. The mixture was incubated overnight at 56 °C with agitation until a homogeneous solution was obtained. The solution was next incubated at 95 °C for 20 min to inactivate proteinase K and then centrifuged at 10,000 × g for 5 min. Genomic DNA was recovered from the supernatant and stored at − 20 °C. Subsequently, the DNA was amplified using Dectin-1 and Dectin-2 primers, as shown in Supplemental Table 1. The PCR products were then run on a 1.5% agarose gel to confirm the mouse genotype.
Induction of colitis with Dextran Sodium Sulfate (DSS)
Mice were given 2% (wt/vol) DSS (molecular weight, 36,000–50,000; MP Biomedicals, Solon, OH) dissolved in drinking water ad libitum for 7 days, followed by a recovery period (water only) of 5 days. Animals were monitored daily for weight loss and disease activity index (including three parameters: weight loss, stool consistency, and presence of blood in feces).
Fecal Microbiota Transfer (FMT)
Microbiota transfer was performed by fecal gavage using a modified version of a previously described protocol [11] as follows: fresh stool samples were recovered from 10 mice (WTj or D-1/2KO mice) and immediately stored in an anaerobiosis generator (Genbox, Biomérieux, Capronne, France) to favor the preservation of anaerobic bacteria. The samples were processed within 6 h in an anaerobic chamber. The feces were rapidly diluted 100-fold in LYHBHI (brain–heart infusion) medium (BD Difco, Le Pont De Claix, France) supplemented with cellobiose (1 mg/ml; Sigma–Aldrich, St. Louis, MO, USA), maltose (1 mg/ml; Sigma–Aldrich), and cysteine (0.5 mg/ml; Sigma–Aldrich). This ready-to-use fecal suspension was used for FMT to mice.
Mice were fasted for 1 h and then subjected to bowel cleansing by oral-gastric gavage with PEG (polyethylene glycol, Macrogol 4000, Fortrans, Ipsen Pharma, France). Four hours later, mice received FMT by intragastric gavage (200 μl of resuspended feces prepared as described above). Mice were then allowed free access to food and water. FMT was repeated twice a week for two weeks before the induction of colitis and continued until the end of the protocol. Bowel cleansing was performed only on day 1.
To exclude the possibility that repeated force feeding of the intestinal microbiota had an effect per se on the phenotype, WTj mice were transplanted with their own fecal microbiota (WTjWTj) and used as the control. Additionally, we chose to use WT mice from Janvier and not cohoused mice to have a completely different microbiota as a baseline.
Gavage with fungi and bacteria
Candida tropicalis ATCC 750 (ATCC, Molsheim, France) and Malassezia restricta CBS7877 (Westerdijk Institute, Utrecht, the Netherlands) were used in this study. C. tropicalis was grown on yeast extract peptone dextrose (YEPD) medium (BD Difco, Le Pont De Claix, France) for 24 h at 37 °C under agitation. M. restricta was grown statically on modified Dixon (mDixon) medium (BD Difco, Le Pont De Claix, France) for 72 h at 34 °C. The cultures were then washed twice in PBS, and a yeast suspension of 107 CFU/mL in 200 μL of PBS or control medium (PBS) was administered daily to mice by intragastric gavage for 7 days before the induction of colitis, and this was continued until the end of the protocol.
Lachnospiraceae strains were also used in the study. Marvinbryantia and Lachnospiraceae bacterium WCA-9-b2 were isolated from the feces of D-1/2KO mice. Briefly, feces from 10 different female D-1/2KO mice were collected in a jar with Genebox Anaer® (Biomerieux, France) to maintain anaerobic conditions. Samples were transferred to an anaerobic chamber and resuspended in pre-reduced complete medium (37 g/L BHI (BD, Difco, USA) + Yeast extract (BD, Difco, USA) 1% + Hemin 5mg/ml (Sigma–Aldrich, St. Louis, MO, USA) + Maltose 0.1% (Sigma–Aldrich, St. Louis, MO, USA) + Cellobiose 0.1% (Sigma–Aldrich, St. Louis, MO, USA) + Cysteine 0.05% (Sigma–Aldrich, St. Louis, MO, USA) + Vitamin K1 0.0001% (Sigma–Aldrich, St. Louis, MO, USA) + Vitamin K3 3.2 μg/ml (Sigma–Aldrich, St. Louis, MO, USA)). They were then successively diluted up to 10−7, and 100 μl of each dilution was spread on agar complete media and incubate for 2 to 3 days at 37 °C in anaerobic conditions. Eighty strains from isolated colonies were sub-cultured in agar complete media and incubate for 2 to 6 days at 37 °C in anaerobic conditions. Then, strains were identified by 16S PCR sequencing.
Blautia hansenii ATCC 27752 was isolated from human feces and provided by ATCC. The bacteria were grown overnight on pre-reduced complete medium (see above) at 37 °C in a static incubator under anaerobic conditions, aliquoted at 5 × 108 CFU/mL and stored at − 80 °C until use. Mice were fasted for 1 h and then subjected to bowel cleansing by oral-gastric gavage with PEG (Macrogol 4000, Fortrans, Ipsen Pharma, France). Four hours later, mice received a bacterial suspension of 108 CFU in 200 μL of PBS or control medium. Bacterial gavage was administered daily to mice by intragastric gavage for 7 days before the induction of colitis, and this was continued until the end of the protocol. Bowel cleansing was only performed on day 1.
Fluconazole treatment
For the antifungal treatment, fluconazole (0.5 mg/mL, Sigma–Aldrich, St. Louis, MO, USA) or control vehicle (PBS) was delivered to mice via drinking water for 7 days before the induction of colitis and continued until the end of the protocol.
Short-chain Fatty Acid (SCFA) administration
Isobutyric acid (0.25 mol/L, Sigma–Aldrich, St. Louis, MO, USA), valeric acid (0.25 mol/L, Sigma–Aldrich, St. Louis, MO, USA), isovaleric acid (0.25 mol/L, Sigma–Aldrich, St. Louis, MO, USA), or control vehicle (PBS) was administered daily to mice by intragastric gavage for 7 days before the induction of colitis, and this was continued until the end of the protocol.
Tissues and samples
Mice were euthanized by cervical dislocation. The distal colon was fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA), and the proximal colon was flushed and frozen for further RNA extraction. Cecal contents were collected and frozen for SCFA quantification. Fecal samples were collected at day 0 and day 7 and at the end of the protocol (day 12) and frozen for gut microbiota analysis and fecal lipocalin level measurements. All samples were stored at − 80 °C until use.
Histology
Colon samples for histological studies were maintained at 4 °C in 4% paraformaldehyde and then embedded in paraffin. Four-micrometer sections (three sections per sample) were stained with hematoxylin and eosin (H&E, Sigma–Aldrich, Saint Louis, USA) and then examined in a blinded manner using a BX43 Olympus microscope to determine the histological score according to previously described methods [12,13,14].
SCFA analysis in cecal samples
Samples were water-extracted, and proteins were precipitated with phosphotungstic acid. A volume of 0.1 μl of the supernatant was analyzed for SCFAs on a gas–liquid chromatograph (Nelson 1020; Perkin-Elmer, St. Quentin en Yvelines, France) equipped with a split-splitless injector, a flame-ionization detector and a capillary column (15 m × 0.53 mm, 0.5 μm) impregnated with SP 1000 (FSCAP Nukol; Supelco, Saint-Quentin-Fallavier, France). The carrier gas (He) flow rate was 10 ml/min, and the inlet, column, and detector temperatures were 175, 100, and 280 °C, respectively. 2-Ethylbutyrate was used as the internal standard. Data were collected and peaks integrated using Turbochrom v6 software (Perkin Elmer, Courtaboeuf, France). Cecal SCFA concentrations are expressed as μmol/g of stool.
Quantification of fecal lipocalin-2 (LCN2) levels
Frozen fecal samples were weighed and suspended in cold PBS. The samples were then agitated on a Precellys (Bertin Corp., France) for 40 s at 5000 rpm with 4.5-mm glass beads to obtain a homogenous fecal suspension. The samples were then centrifuged for 5 min at 10,000 × g (4 °C), and clear supernatants were collected and stored at − 20 °C until analysis. LCN2 levels were estimated using a DuoSet murine LCN2 ELISA kit (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions and expressed as ng/mg of stool.
RNA extraction and gene expression analysis using quantitative real-time PCR (qRT–PCR)
Total RNA was isolated from colon samples using an RNeasy Mini Kit (Qiagen, Hilden, Germany), including a DNAse treatment step, according to the manufacturer’s instructions. Quantitative RT–PCR was performed using a Luna® Universal One-Step RT–qPCR Kit (New England Biolabs, Massachusetts, USA) followed by qPCR using Luna® Universal qPCR Master Mix (New England Biolabs, Massachusetts, USA) in a StepOnePlus apparatus (Applied Biosystems, Foster City, CA, USA) with specific mouse oligonucleotides. Amplification was initiated with an enzyme activation step at 95 °C for 10 min, followed by 40 cycles consisting of a 15 s denaturation step at 95 °C and a 60 s annealing step at 60 °C and a melting curve consisting of a step of temperature increase from 60 °C to 95 °C with a fluorescence analysis every 0.3 s. The primer sequences of the amplified target are listed in Supplemental Table 1. We used the 2−ΔΔCt quantification method with mouse GAPDH as a control.
Fecal DNA extraction
Fecal total DNA was extracted from weighed stool samples as previously described [15], with modifications. After nucleic acid precipitation with isopropanol, DNA suspensions were incubated overnight at 4 °C and centrifuged at 20,000 × g for 30 min. The supernatants were transferred to a new tube containing 2 μL of RNase (RNase A, 10 mg/ml; EN0531; Fermentas, Villebon sur Yvette, France) and incubated at 37 °C for 30 min. Nucleic acids were precipitated by the addition of 1 ml of absolute ethanol and 50 μL of 3 M sodium acetate and centrifuged at 20,000 × g for 10 min. The DNA pellets were washed with 70% ethanol 3 times, dried and resuspended in 100 μl of Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, adjusted pH 8). The DNA suspensions were stored at − 20 °C for real-time qPCR analysis of the 16S rDNA or ITS2 sequences.
Fungal and bacterial quantification via quantitative PCR (qPCR)
Fecal extracted DNA was subjected to qPCR by using Luna® Universal qPCR Master Mix (New England Biolabs, MA, USA) for quantification of all fungal sequences or by using TaqMan Gene Expression Assays (Life Technologies) for quantification of all bacterial sequences. For all fungal quantification, amplification was initiated with an enzyme activation step at 95 °C for 5 min, followed by 45 cycles consisting of a 15 s denaturation step at 94 °C, a 30 s annealing step at 55 °C, and a 30 s elongation step at 72 °C. This was followed by a single step of 5 min at 72 °C and a melting curve consisting of a step of temperature increase from 60 °C to 95 °C with a fluorescence analysis every 0.3 s. For all bacterial quantification, amplification was initiated with an enzyme activation step at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles consisting of a 15 s denaturation step at 95 °C and a 60 s annealing/extension step at 60 °C. The probes and primers for the bacterial and fungal genes are listed in Supplemental Table 1. We used the 2−ΔΔCt quantification method with fecal weight and calibrated the assay to the control group.
16S DNA gene and ITS2 sequencing
Bacterial diversity was determined for each sample by targeting a variable portion of the ribosomal genes. PCR was performed to prepare amplicons using V3-V4 oligonucleotides (PCR1F_460: 5′ CTTTCCCTACACGACGCTCTTCCGATCTACGGRAGGCAGCAG 3′, PCR1R_460: 5′ GGAGTTCAGACGTGTGCTCTTCCGATCTTACCAGGGTATCTAATCCT 3′). Amplicon quality was verified by gel electrophoresis, and the amplicons were sent to the @BRIDGe platform for sequencing on an Illumina MiSeq (Illumina, San Diego, CA, USA).
A similar approach was used for the fungal microbiota using the ITS2 primers 5′-GTGARTCATCGAATCTTT-3′ (sense) and 5′-GATATGCTTAAGTTCAGCGGGT-3′ (antisense) and the optimized and standardized ITS2 amplicon library preparation protocol (Metabiote, GenoScreen).
16S and ITS2 sequence analysis
For 16S sequences, the sequences were demultiplexed and quality filtered using the QIIME2 version 2021.2.0 software package [16]. The sequences were then assigned to OTUs using the UCLUST algorithm [17] with a 97% pairwise identity threshold and classified taxonomically using the Greengenes reference database (version 13.8) [18]. Rarefaction was performed on both datasets and used to compare the relative abundances of OTUs across samples. Alpha diversity was estimated using the Shannon diversity index or the number of observed species. Beta diversity was measured by a Jaccard distance matrix and was used to build principal coordinates analysis (PCoA) plots. The linear discriminant analysis (LDA) effect size (LEfSe) algorithm was used to identify taxa that were specific to a genotype [19].
For ITS2 sequences, data were processed using the FROGS pipeline (available from http://frogs.toulouse.inra.fr), established in Toulouse, France [20], for sequence quality control, filtering, and affiliation of taxa. The sequences were assigned to OTUs with a 97% threshold of pairwise identity and classified taxonomically using the UNITE ITS database (version 8_2) [21]. The phyloseq package for R was used for alpha and beta diversity analyses as well as all illustrations. The Deseq2 package for R was used for differential analysis of OTUs with respect to the different phenotypes [22]. The LEfSe algorithm was used to identify taxa that were specific to genotype.
Deposition of the raw sequence data in the SRA database from the NCBI, the accession numbers are the following: PRJNA824403 and PRJNA824345.
Bone marrow-derived dendritic cell (BMDC) preparation and stimulation
Primary cultures of BMDCs were obtained from WT, D-1KO, D-2KO, and D-1/2KO mice as previously described [23] with minor modifications. Briefly, bone marrow cells isolated from mouse tibiae and femurs were washed with Roswell Park Memorial Institute (RPMI) 1640 and passed through 40 μm cell filters (Falcon, USA). Cells were then cultured and differentiated in petri dishes in 10 mL of RPMI 1640 with 10% (vol/vol) FCS (Eurobio Scientific), 100 U/mL penicillin, 100 μg/mL streptomycin (Sigma–Aldrich) and 20 ng/mL murine granulocyte-macrophage colony-stimulating factor (GM-CSF Peprotech, Germany) for 9 days at 37 °C and 5% CO2.
On day 9, immature BMDCs were harvested and seeded into 96-well plates at 105 cells/well. The cells were stimulated with bacterial strains (1:5 cell-to-bacteria ratio) killed by UV, lipopolysaccharide (LPS) (150 ng/mL), depleted zymosan (100 μg/mL, InvivoGen), and yeast mannan (100 μg/mL, Sigma–Aldrich) overnight at 37 °C. The culture supernatants were collected, and the levels of IL-6 and TNFα were measured by ELISA kits (Thermo Fisher Scientific) according to the manufacturer’s protocol.
Statistical analysis
GraphPad Prism version 7 (San Diego, CA, USA) was used for all analyses and preparation of graphs. For all data displayed in graphs, the results are expressed as the mean ± SEM (n = 4 to 16 per group). For comparisons between two groups, a two-tailed Student’s t test for unpaired data or a nonparametric Mann–Whitney test was used. For comparisons among more than two groups, one-way analysis of variance (ANOVA) and a post hoc Tukey test or a nonparametric Kruskal–Wallis test followed by a post hoc Dunn’s test were used. For comparisons with multiple factors, two-way ANOVA and a post hoc Tukey test were used. For all statistical tests, differences with a p value less than 0.05 were considered to be statistically significant: *p < 0.05, **p < 0.01, ***p < 0.001.