Interferon regulatory factor 1 (IRF-1) promotes intestinal group 3 innate lymphoid responses during Citrobacter rodentium infection

Animals and housing conditions

Irf1–/– mice45 were kindly provided by A. Kröger (University of Magdeburg). Irf1–/– mice were bred with Irf1+/– as littermate controls or were co-housed with Irf1+/+ mice for at least four weeks to ensure adaptation of the intestinal microbiome. No evident differences in α and β-diversity of the intestinal microbiota could be detected by 16S-based metagenomic sequencing (Supplementary Fig. 6A, B). Ifng-/- mice were originally purchased from the Jackson Laboratory and were bred in-house. Irf1fl/fl mice were generated in-house through crossing a mouse strain purchased from the European Mouse Mutant Archive (EMMA) of the European Conditional Mouse Mutagenesis Program (EUCOMM) with a general FLP deleter strain. Irf1fl/fl mice were crossed to Tie2-cre mice or Villin-cre mice (Jackson Laboratory) to generate Irf1ΔTie2 and Irf1ΔIEC mice respectively. All mice were bred on a C57BL/6 background and kept in individually ventilated cages. Mice of different experimental groups were age- and sex-matched. Sterile drinking water and food were provided ad libitum. Animal husbandry and experimental procedures were approved by the Government of Unterfranken (55.2.2-2532-2-712).

C. rodentium infection model and quantification of the bacterial burden

To analyze the relevance of IRF-1 during infectious colitis, mice were infected with an erythromycin resistant and luminescent strain of Citrobacter rodentium (C. rodentium; strain ICC169) kindly provided by Christian Riedel46. Prior to infection, mice were fasted for 8 hours. C. rodentium was cultivated in sterile, erythromycin-supplemented (500 µg/ml) LB-medium at 37 °C with shaking and used for infection during the phase of exponential growth. For infection, bacteria were resuspended in sterile PBS and mice were inoculated with ~4 × 109 CFU of C. rodentium in 200 µl PBS by oral gavage using a feeding needle. For analysis of bacterial burdens, C. rodentium luminescence was measured by in vivo-imaging using an IVIS Spectrum CT system (PerkinElmer, Waltham, Massachusetts). Therefore, the abdomen of the infected mice was depilated and they were anesthetized with 1,5-2% isoflurane. Quantification of luminescence was performed using the IVIS-associated software Living Image 4.0. For determination of C. rodentium in feces, fresh stool samples were collected and weighed. To quantify C. rodentium CFUs in liver, spleen or mLN, fresh tissue samples were weighed, covered with LB medium (1 ml LB medium per 0.1 g tissue) and homogenized in a mixer mill (MM 400, Retsch, Germany) at a frequency of 25 Hz for 2 min. Serial dilutions of dissolved stool pellets, blood or tissue homogenates were plated on erythromycin-supplemented LB-Agar plates. After an incubation time of 20 h at 37 °C, C. rodentium colonies were counted.

Bone marrow chimeric mice

For the generation of bone marrow chimeric mice, lethally irradiated (10 Gray) C57BL/6 mice or congenic B6.SJL mice were reconstituted with 1 × 107 femoral bone marrow cells of either Irf1+/+ mice or Irf1-/- mice via i.v. injection. In some experiments, Rag2–/–γc–/– were irradiated with a dose of 5 Gray and i.v. transferred with mixed donor bone marrow containing 80% Rorgt–/– and 20% wildtype or Irf1–/– bone marrow cells. Reconstituted mice were treated with antibiotics (Borgal, Virbac, France) for 2–3 weeks to prevent infections in the recovery phase. Eight weeks after hematopoietic reconstitution, bone marrow chimeric mice were analyzed in the steady state or infected with C. rodentium.

Isolation of lymphocytes from the lamina propria and from mesenteric lymph nodes

Single cell suspensions from mesenteric lymph nodes were prepared through digestion with Collagenase B (0.25 mg/ml; Roche) and DNase I (0.05 mg/ml Roche) using a gentleMACS Octo Dissociator (program: 37c_m_SDK_1; Miltenyi Biotec) according to the manufacturer’s recommendations. For the isolation of lamina propria mononuclear cells (LPMCs) intestinal tissue was removed and cleaned from residual fat. Luminal contents were flushed out and the intestinal tissue was cut longitudinally and then laterally into pieces of 5 mm length. LPMCs were isolated with the lamina propria dissociation kit mouse from Miltenyi Biotec according to the manufacturer’s instructions under use of a gentleMACS Octo Dissociator (Miltenyi Biotec) running the program m_intestine_01. After the isolation process, the cell suspension was proceeded to Percoll gradient centrifugation (40%/80%) for purification. Epithelial cells for bulk RNAseq analysis were isolated by incubating longitudinally opened and cleaned intestinal tissue in PBS with 1 mM DTT for 10 min and subsequently in 20 ml of prewarmed HBSS with 1.5 mM EDTA for 15 min. Next, the tissue was vortexed for 1–2 min at maximum speed and intraepithelial lymphocytes were removed using Percoll gradient centrifugation (40%/80%). The pellet containing intestinal epithelial cells was centrifuged for 10 min at 400 g and the pellet was immediately subjected to RNA extraction.

Flow cytometry

Prior to staining with antibodies against specific intra- or extracellular markers, freshly isolated LPMCs were incubated with anti-CD16/CD32 antibodies (anti-Fc-receptor; clone 93; eBioscience) for 10 min at 4 °C to block unspecific binding. To distinguish between cells of the innate or the adaptive compartment, LPMCs were incubated with a cocktail of biotinylated lineage antibodies including anti-B220 (RA3-6B2; eBioscience, 1:50), anti-CD3 (REA641, 1:50), anti-CD5 (1:50), anti-GR1 (RB6-8C5; eBioscience, 1:50), anti-SiglecF (REA798, 1:50) and anti-Ter119 (Ter-119, 1:50) for 10 min at 4 °C. After washing, streptavidin conjugated Brilliant Violet 421 (BioLegend, 1:666) or VioBright FITC (Miltenyi Biotec, 1:100) was applied for 30 min at 4 °C in a secondary staining combined with a selection of the following antibodies used for surface staining: anti-Thy1.2 (CD90.2 in PerCP-Vio700, 1:20), anti-CCR6 (CD196 in PE, 1:20), anti-NKp46 (REA815 in FITC, 1:50), anti-Eomes (REA116 in PE, Miltenyi, 1:10), anti-NK1.1 (PK136 in BV 421, 1:20) anti-IL23R Ab (FAB16861R in AF 647, R&D Systems, 1:66) and anti-IL-12Rβ1 (CD212 in PE; BD Pharmingen, 1:20). Subsequently, cells were fixed and permeabilized using the FoxP3 Transcription Factor Staining Buffer Set (Invitrogen) according to the manufacturer’s instructions followed by intracellular transcription factor staining with fluorochrome-coupled anti-RORγt (Q31-378 in AlexaFluor 647 or BV421; BD Pharmingen, 1:80), anti Gata3 (REA174, Miltenyi Biotec, 1:10) and anti-Tbet (eBio4B10 in PE; eBioscience, 1:40) antibodies (30 min at 4 °C). For intracellular cytokine measurements, anti-IL-22 (Poly5164 in PE; BioLegend, 1:20), anti-IL-17A (TC11-18H10.1 in PE/Cyanine7; BioLegend, 1:20) and anti-IFN-γ (XMG1.2 APC; eBioscience, 1:158) antibodies were applied in combination with the transcription factor staining. In this case, cells were stimulated with Cell Stimulation Cocktail plus protein transport inhibitors (eBioscience) according to manufacturer’s instructions for 4 h prior to antibody staining. Antibodies for flow cytometry were purchased from Miltenyi Biotech, unless specified otherwise. Samples were measured on an LSRFortessa cell analyzer (BD Biosciences, FacsDiva Software version 6.1.3) and evaluated with FlowJo 10 (FlowJo LLC). The gating strategy for characterization of the various immune cell populations is outlined in Supplementary Fig. 7.

ILC sort

To purify intestinal ILCs for analysis in cell culture or for RNASeq analysis, we performed fluorescence activated cell sorting using a MoFlo Astrios EQ device (Beckman Coulter) within the Core Unit Cell Sorting Erlangen. ILCs were purified using the following surface marker panel: CD45+ (REA737 in VioBlue, 1:50), B220 (REA755 in FITC, 1;50), CD3 (REA641 in FITC, 1:50), CD5 (in FITC, 1:50), CD11b (REA592 in APC-Vio770, 1:50), CD11c (REA754 in APC-Vio770, 1:50), KLRG1 (2F1 in APC; eBioscience, 1:158), CD127+ (IL-7Rα; A7R34 in PE; BioLegend, 1:20), Thy1.2+ (in PerCP-Vio700, 1:20). Unless specified otherwise, all antibodies used for cell sorting were purchased from Miltenyi Biotec.

In vitro ILC stimulation

To assay the cytokine responses of ILC3s ex vivo, ILCs were sort purified. Therefore, freshly isolated single cell suspensions from ileum, colon and mLNs of two mice of either Irf1+/– or Irf1–/– genotype were pooled and stained prior to cell sorting. For in vitro stimulation, 2 × 104 sorted CD45+ B220 CD3 CD5 CD11b CD11c KLRG1 CD127+ Thy1.2+ cells were plated in 200 µl DMEM GlutaMAX medium (Gibco) supplemented with 10% FBS (Gibco), 1× MEM nonessential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 20 mM Hepes (Carl Roth), 50 µM 2-mercaptoethanol (Sigma-Aldrich) and 1 % penicillin-streptomycin (Sigma-Aldrich) in the presence of 20 ng/ml recombinant mouse IL-1β (ImmunoTools) and 20 ng/ml recombinant mouse IL-23 (Miltenyi Biotec). After 24 h or 72 h, culture supernatants were harvested for determination of cytokine concentrations by ELISA and residual ILCs were lysed for isolation of RNA.

Gene expression analysis

Tissue pieces from distal colon or ileum were snap-frozen in liquid nitrogen and stored at −80 °C for RNA isolation. RNA was isolated from tissue using the NucleoSpin RNA Plus kit (#740984, Macherey-Nagel) according to the manufacturer’s recommendations. To analyze gene expression of sort purified ILC3s, we used the peqGOLD Micro Spin Total RNA Kit (#13-6831, VWR) following the manufacturer’s instructions. cDNA was synthesized with the Script RT-PCR kit (#PCR-511L, Jena Bioscience, Germany). Gene expression was analyzed via quantitative real-time PCR (qRT-PCR) using pre-designed QuantiTect Primer assays (Qiagen) in a CFX Connect System (Bio-Rad). ΔCT values were calculated to illustrate the expression of the indicated genes relative to the housekeeping gene hypoxanthine phosphoribosyltransferase 1 (hprt).

Bulk RNAseq

To analyze total transcriptome profiles of whole colon tissue (Fig. 2a–c) after C. rodentium infection, we isolated total RNA from the distal colon of Irf1+/+ and Irf1-/- mice 9 d after infection. For applying the NucleoSpin RNA Plus kit (Macherey-Nagel). A total amount of 2 μg RNA per sample was used for the generation of sequencing libraries using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) following manufacturer’s instructions. Library preparations were sequenced on an Illumina platform and paired-end reads were generated. Paired-end clean reads were mapped to the reference genome (mm10) using HISAT2 (v.2.0.5) software with default parameters47. FeatureCounts (v.1.6.4) was used to count the read numbers mapped to each gene48. Differential expression analysis between two conditions with 4 biological replicates per condition was performed using DESeq2 (v.1.22.2)49. The resulting p values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR). Genes with an adjusted p value < 0.05 found by DESeq2 were assigned as differentially expressed.

Cytokine measurements by Enzyme-linked immunosorbent assay (ELISA)

To quantify the concentrations of IL-22, IFN-γ and IL-17A in sera and cell supernatants, Ready-SET-Go ELISA Sets from eBioscience were used according to the manufacturer’s instructions.

16 S Next generation sequencing of fecal microbiota

Fecal samples were collected and immediately stored at −80 °C. Genomic bacterial DNA was isolated with the PSP Spin Stool DNA Kit (Stratec molecular, Germany) according to manufacturer’s recommendations. 10 ng of DNA was used to amplify the V3-V4 region of bacterial 16sRNA genes using the NEBNext Q5 Hot Start HiFi PCR Master Mix (NEB). Amplified PCR products were isolated with AMPure XP Beads (Beckmann Coulter Genomics), purified, pooled and subjected to 2 × 300 bp paired-end sequencing on an Illumina MiSeq platform50.


For immunohistochemical (IHC) stainings, distal colonic tissues from mice infected with C. rodentium were fixed in buffered formalin (Roti-Histofix; Carl Roth) at 4 °C for 24 h, dehydrated, and embedded in liquid paraffin. 3-µm sections were cut using a microtome (Leica) and processed for IHC applying the Tyramide Signal Amplification (TSA) Cy3 system (Perkin Elmer) according to the manufacturer’s protocol. To analyze the expression level of IRF-1, a primary antibody from Cell Signaling (D5E4) was used (1:50 dilution). To visualize the colonization of the mucosal surface with C. rodentium, a primary antibody from Abcam (ab37056, 1:1000) was applied. Both primary antibodies were used in combination with a goat-anti-rabbit biotinylated secondary antibody (Jackson Immuno Research). Epithelial cells were stained with Alexa Fluor 488 anti-mouse CD326 (Ep-CAM; G8.8; BioLegend, 1:100). Nuclei were counterstained with DAPI (Invitrogen). Pictures were acquired on a Leica TCS SP5 II confocal microscope using Leica LAS AF version software.

Multiphoton microscopy

Infected animals were sacrificed and fresh tissue samples from distal colons were kept in ice-cold PBS, and multiphoton microscopy (MPM) was performed on the same day. In total, 42 3D image stacks (nKO = 18, nhet = 24) were recorded and analysed from six animals (n = 3/group). An upright Multiphoton microscope (TriMScope II, LaVision BioTec, Bielefeld, Germany) was used in combination with a water immersion objective (HC Fluortar L 16x/0.6 W VISIR, Leica microsystems, Wetzlar, Germany) and a femtosecond-pulsed Ti-Sapphire laser (Chameleon Vision II, Coherent, Santa Clara, CA, USA), at a wavelength of 810 nm. Exponential z adaptation of the laser power was used to compensate for attenuation at greater tissue depths. As previously described, the optical filters were chosen to target second harmonic generation (SGH) from collagen-I (ET405/20, Chroma, Vermont, USA), and natural autofluorescence from NADH in mucosal epithelial cells (450/70 BrightLine HC, Semrock Inc., Rochester, NY, USA)17. In addition to that, a third channel was used to target fluorescence from mRuby-expressed by the reporter bacteria. MPM stacks were recorded at an axial spacing of 2 µm. The lateral image size was 682 × 682 µm², separated into 512 × 512 or 1024 × 1024 pixels. The image contrast was adjusted manually using Fiji/ImageJ 1.5 upon visual inspection. No further image processing has been used. The number of bacterial clusters was counted manually in each image and divided by the area of the field of view in order to obtain the cluster density. The average cluster density per sample was calculated from all six images of the respective sample.


Experimental results were plotted and analyzed for statistical significance with GraphPad Prism 8.3 (GraphPad Software Inc.), Excel 2016 and R 4.2.0. Data are shown as mean ± SEM. For comparison of two independent experimental groups, a two-tailed Mann–Whitney U test was used. If more than two groups were compared, one-way ANOVA (Kruskal Wallis Test) with Dunnett´s multiple comparisons test or 2way ANOVA with Tukey´s multiple comparisons test was performed. Differences of p ≤ 0.05 were considered as statistically significant indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; *** p ≤ 0.001; ****p ≤ 0.0001).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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