Interleukin 10 controls the balance between tolerance, pathogen elimination, and immunopathology in birds
- The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, United Kingdom
- The Royal Veterinary College, United Kingdom
Figures
Figure 1 with 4 supplements
IL10 knockout (IL10KO) and IL10-Enhancer knockout (IL10EnKO) editing strategies.
(A) Schematic of the pipeline used to create gene-edited chickens using embryonic primordial germ cells (PGCs) and the iCaspase9 sterile surrogate host line. Male PGCs carrying the desired biallelic (homozygous [HOM]) edits were injected into iCaspase9 sterile surrogate host embryos to create IL10KO and IL10EnKO chickens. Upon sexual maturity, surrogate host cockerels were bred to RIR hens, resulting in 100% IL10KO heterozygous (HET) or IL10EnKO HET offspring in the first generation (G1). (B) Schematic of the IL10 locus depicting the IL10 putative enhancer region and the five IL10 exons (Ex1 to Ex5). (C) IL10EnKO HOM PGCs were created by deleting a 533 bp fragment encompassing the IL10 putative enhancer region, using two guide RNAs (gRNAs); DNA sequencing confirmed the biallelic deletion. (D) IL10KO HOM PGCs were created using one gRNA and a 143 bp repair template (ssODN) to modify three nucleotides in IL10 exon 1 (red, lowercase), thus introducing a premature in-frame stop codon in the IL10 gene (underlined, *); DNA sequencing confirmed the biallelic edit.
Figure 1—figure supplement 1
Wild-type and edited IL10 sequences for IL10 knockout (IL10KO) edit.
(A) Full-length chicken IL10 protein sequence (blue; 175 amino acids; NP_001004414.2) aligned to IL10 coding sequence. Alternating exons are shown in black and green; the start codon is underlined; the stop codon is shown in red (*). (B) Chicken IL10 exon 1 sequence (black), showing the sequence and position of guide RNA (gRNA) ggIL10_exon1_g3 (green box), Protospacer Adjacent Motif (PAM; yellow box), and ssODN IL10_exon1_HDRoligo2 (grey box), with the three substituted nucleotides (red, lowercase). (C) Edited chicken IL10 exon 1 showing the premature stop codon in red (*) and truncated IL10 peptide (25 amino acids).
Figure 1—figure supplement 2
Wild-type and edited IL10 putative enhancer sequences for IL10-Enhancer knockout (IL10EnKO) edit.
The chicken IL10 putative enhancer region (548 bp) is shown in blue, with the core conserved region (Figure 1—figure supplement 3) double-underlined. The turquoise and pink boxes highlight the location of the AP1 and FOXP3 sites, respectively. The position and sequence of guide RNAs (gRNAs) IL10_enhancer_guide1 and IL10_enhancer_guide2 and corresponding Protospacer Adjacent Motifs (PAMs) are indicated by green and yellow boxes, respectively. The 533 bp sequence deleted in IL10EnKO primordial germ cells (PGCs) is indicated by a grey box.
Figure 1—figure supplement 3
Alignment of conserved enhancer-like sequences in the IL10 locus of different avian species.
A 6 kb genomic DNA sequence upstream of the first exon of the chicken IL10 locus (Position Chr 26;2,564,995, GRCg6a:CM000117.5) was used to identify conserved sequences in multiple avian genomes using BLAST. A single highly conserved region was located around 2.3–2.8 kb upstream of the IL10 transcription start site in each species shown. The sequences were aligned using ClustalW. Candidate purine-rich binding sites for ETS/NFAT family transcription factors (red), AP1 (turquoise box), and FOXP3 (pink box) are indicated.
Figure 1—figure supplement 4
Genotyping strategy for IL10 knockout (IL10KO) and IL10-Enhancer knockout (IL10EnKO) edited primordial germ cells (PGCs) and birds.
(A) Primers IL10_Exon1_F4 and IL10_Exon1_R4 were used to amplify a 1826 bp fragment encompassing IL10 exon 1 (uncut), followed by digestion with AvrII (cut) to identify PGC clones carrying the IL10KO edited allele. (B) Primers IL10_Exon1_F1 and IL10_Exon1_R1 were used to amplify a 246 bp fragment encompassing IL10 exon 1, followed by digestion with AvrII to identify birds carrying the IL10KO edited allele (gel shows PCR products after AvrII digestion). (C) Primers IL10-Enhancer_F2 and IL10_Enhancer_R2 were used to amplify the IL10 putative enhancer region and identify PGC clones carrying the IL10EnKO edited allele. (D) Primers IL10-Enhancer_F2 and IL10_Enhancer_R2 were also used to identify birds carrying the IL10EnKO edited allele. See Supplementary file 3 for primer sequences and expected fragment sizes.
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Figure 1—figure supplement 4—source data 1
PDF file containing the annotated raw, unedited, and uncropped agarose gel images shown in Figure 1—figure supplement 4, with the relevant bands clearly labelled.
- https://cdn.elifesciences.org/articles/106252/elife-106252-fig1-figsupp4-data1-v1.zip
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Figure 1—figure supplement 4—source data 2
Original files of the raw, unedited, and uncropped agarose gel images shown in Figure 1—figure supplement 4.
- https://cdn.elifesciences.org/articles/106252/elife-106252-fig1-figsupp4-data2-v1.zip
Figure 2
Confirmation of the IL10 mutation in bone marrow-derived macrophages (BMDMs).
(A) IL10 protein levels were measured by capture ELISA for lipopolysaccharide (LPS)-stimulated BMDMs derived from day 18 IL10 knockout (IL10KO) wild-type (WT) (n=4), HET (n=2), homozygous (HOM) (n=4), and IL10-Enhancer knockout (IL10EnKO) HOM (n=4) embryos. Two independent experiments were performed, and protein levels were normalised against the highest IL10KO WT value for each experiment and then combined. LPS treatment induced IL10 production in IL10KO WT BMDMs, but not in IL10KO HOM BMDMs; intermediate levels of IL10 were detected in IL10KO HET and IL10EnKO HOM BMDMs; IL10 expression in non-LPS-induced samples was negligible (<0.015; not shown). (B) Nitric oxide production was assessed by measuring nitrite levels using Griess assay for LPS-stimulated BMDMs derived from day 18 IL10KO WT (n=6), HET (n=2), HOM (n=7), and IL10EnKO HOM (n=6) embryos, in the absence or presence of neutralising anti-IL10 antibody ROS-AV163 (anti-IL10). Three independent experiments were performed and nitrite levels were normalised against the highest IL10KO HOM nitrite value for each experiment before being combined. A significant increase in nitrite levels was observed in the absence of IL10 in LPS-stimulated IL10KO HOM BMDMs; nitrite levels were also significantly increased in LPS-stimulated IL10KO HET and IL10EnKO HOM BMDMs. Addition of neutralising anti-IL10 antibody did not significantly affect nitrite levels in LPS-stimulated BMDMs, independently of the IL10 genotype. Note that each BMDM sample was derived from three pooled embryos of the same genotype. Data displayed as mean with SE. Statistical significance calculated using two-tailed unpaired t tests; *p<0.05, **p<0.01, ***p<0.001, ns: not significant.
Figure 3
Growth curves and histopathological analyses of the gastrointestinal tract of IL10-deficient chickens in specified pathogen-free (SPF) and conventional facilities.
(A) Growth curves for IL10 knockout (IL10KO) wild-type (WT) (n=3–8), heterozygous (HET) (n=11–16), and homozygous (HOM) (n=2–4) hens raised in the National Avian Research Facility (NARF) SPF facility, from hatch to 14 weeks. No significant weight differences were observed between genotypes at any timepoint (p>0.05). (B) Growth curves for IL10-Enhancer knockout (IL10EnKO) WT (n=2–4), HET (n=7–11), and HOM (n=5–11) hens raised in the NARF SPF facility, from hatch to 24 weeks. IL10EnKO HET and HOM hens were overall heavier than WT controls, but this was statistically significant only between day 8 and day 24 (p<0.05). (C) Sums of histopathological scores for IL10KO WT (n=5) and HOM (n=9), and for IL10EnKO WT (n=9) and HOM (n=11) birds raised in the NARF SPF facility; tissue samples were collected at regular intervals from 16 to 40 weeks post-hatch. No significant differences were observed between genotypes for the tissues analysed. (D) Growth curves for IL10KO WT (n=2–10) and HOM (n=5–15) hens raised in the NARF conventional facility, from hatch to 48 weeks. IL10KO HOM hens were significantly smaller than WT controls from 3 to 19 weeks post-hatch (p<0.05), but this difference resolved with age. (E) Sums of histopathological scores for IL10KO WT and HOM birds (n=11 in each group) raised in the NARF conventional facility; tissue samples were collected at regular intervals from 10 to 50 weeks post-hatch. No significant differences were observed between genotypes for the tissues analysed. (F) Cellular infiltration scores for IL10KO WT and HOM birds raised in the NARF SPF and conventional facilities (same bird numbers as in C and E). Cellular infiltration scores were overall significantly higher for IL10KO WT and HOM birds raised in the conventional facility. Maximum possible sums of scores for data in C and E=11; score range for data in F=0–3. Data displayed as mean with SD (A, B, D) or as median with 95% confidence interval (C, E, F). Statistical significance calculated using one-way ANOVA with Bonferroni multiple comparison tests (A, B), Kruskal-Wallis test (C), two-tailed unpaired t tests (D), or Mann-Whitney U tests (E, F); *p<0.05, **p<0.01, ns: not significant.
Figure 4
Impact of IL10 knockout (IL10KO) homozygous (HOM) mutation on the caecal microbiota of chickens.
(A–B) Sequencing of 16S rDNA variable regions amplified from DNA extracted from the caecal contents of IL10KO wild-type (WT) and HOM birds (n=5 in each group) at 4 weeks of age revealed differences in the relative abundance of microbial phyla (A) and genera (B). Each vertical bar shows data from a different bird. (C) Linear discriminant analysis (LDA) effect size (LefSe) analysis identified differentially abundant taxa between IL10KO WT and HOM birds. (D) Principal component analyses based on weighted Unifrac distance showed clear clustering of samples (Adonis2 R2=0.25, p=0.017). Axis 1: first principal component, Axis 2: second principal component; value in brackets: total percentage of variation between samples.
Figure 5
Response to vaccination in IL10 knockout (IL10KO) wild-type (WT) and homozygous (HOM) chickens.
Blood samples were collected from 29-week-old IL10KO WT and HOM vaccinated hens raised in the National Avian Research Facility (NARF) conventional facility (n=8 in each group), and antibody titres were measured by ELISA. Titres to avian encephalomyelitis virus (AEV) and infectious bursal disease virus (IBDV) were significantly lower in IL10KO HOM hens compared to WT controls, whereas titres to duck adenovirus (the agent of egg drop syndrome [EDS]) and infectious bronchitis virus (IBV) were significantly higher in IL10KO HOM hens compared to WT controls; titres to chicken anaemia virus (CAV), infectious laryngotracheitis virus (ILTV), and Newcastle disease virus (NDV) were not significantly different between IL10KO HOM and WT hens. Data displayed as median with 95% confidence interval. Statistical significance calculated using Mann-Whitney U tests; *p<0.05, **p<0.01, ns: not significant.
Figure 6 with 5 supplements
Net replication of C. jejuni and S. Typhimurium in the caeca of IL10 knockout (IL10KO) wild-type (WT), heterozygous (HET), and homozygous (HOM) chickens and immune responses to infection.
(A) Caecal burden of C. jejuni strain M1 in IL1KO WT, HET, and HOM chickens at 1 and 2 weeks post-inoculation in two separate trials. (B) Heat map of differentially transcribed genes measured by multiplex quantitative reverse transcriptase-PCR (qRT-PCR) in the caeca of IL10KO HOM chickens in relation to IL10KO WT birds at 1 and 2 weeks post-inoculation in two separate trials. An increase in the expression of pro-inflammatory genes was consistently observed in both studies. (C) Burden of S. Typhimurium strain ST4/74 in the caeca, liver, and spleen of IL10KO WT, HET, and HOM chickens at 1 and 2 weeks post-inoculation (single trial). (D) Heat map of differentially transcribed genes in the caeca of IL10KO WT and HOM chickens 1 and 2 weeks post-inoculation with ST4/74. All group sizes: n=10 or 11. Bacterial colonisation shown as median with 95% confidence interval; dotted line shows limit of detection for each study (A, C). Statistical significance calculated using one-way ANOVA with Kruskal-Wallis test followed by Dunn’s multiple comparison tests; *p<0.05, **p<0.01, ***p<0.001, ns: not significant.
Figure 6—figure supplement 1
Histopathological findings in the ileum and caecum of IL10 knockout (IL10KO) wild-type (WT), heterozygous (HET), and homozygous (HOM) chickens infected with C. jejuni cohort 1.
Histological sections from the ileum (A) and caecum (B) at 1 week post-infection, and from the ileum (C) and caecum (D) at 2 weeks post-infection were scored blind according to the scoring system described in Materials and methods, by evaluating the extent of lymphocyte, plasma cell, and heterophil infiltration. p-Values are shown where statistical differences between groups were detected.
Figure 6—figure supplement 2
Histopathological findings in the ileum and caecum of IL10 knockout (IL10KO) wild-type (WT), heterozygous (HET), and homozygous (HOM) chickens infected with C. jejuni cohort 2.
Histological lesions from the ileum (A) and caecum (B) at 1 week post-infection, and from the ileum (C) and caecum (D) at 2 weeks post-infection were scored in an identical fashion to cohort 1 (Figure 6—figure supplement 1).
Figure 6—figure supplement 3
Histopathological findings in the ileum and caecum of IL10 knockout (IL10KO) wild-type (WT), heterozygous (HET), and homozygous (HOM) chickens infected with S. Typhimurium.
Histological lesions from the ileum (A) and caecum (B) at 1 week post-infection, and from the ileum (C) and caecum (D) at 2 weeks post-infection were scored in an identical fashion to Campylobacter infection (Figure 6—figure supplement 1).
Figure 6—figure supplement 4
Differential gene expression in the caecum of IL10 knockout (IL10KO) wild-type (WT) and homozygous (HOM) chickens infected with C. jejuni.
Volcano plots showing the direction and magnitude of changes in the transcription of immunity-related genes in the caeca of IL10KO WT versus IL10KO HOM chickens following infection with C. jejuni in cohort 1 at 1 week (A) and 2 weeks (B) post-infection, and in cohort 2 at 1 week (C) and 2 weeks (D) post-infection.
Figure 6—figure supplement 5
Differential gene expression in the caecum of IL10 knockout (IL10KO) wild-type (WT) and homozygous (HOM) chickens infected with S. Typhimurium at 1 week (A) and 2 weeks (B) post-infection.
Analysis of differential transcription was identical to that of Campylobacter infection (Figure 6—figure supplement 4).
Figure 7
E. tenella replication and impact on host pathology and performance in wild-type (WT), heterozygous (HET), and homozygous (HOM) G2 populations of IL10 knockout (IL10KO) and IL10-Enhancer knockout (IL10EnKO) chickens.
(A, B) Parasite replication calculated as parasite genomes per host genome detected by quantitative PCR in genomic DNA extracted from caecal tissue 6 days post-infection (dpi). (C, D) Haematoxylin and eosin stained caecal tissues collected from IL10KO WT and HOM chickens 6 days post E. tenella infection. Numerous gametocytes are visible in the IL10KO WT line (C); gametocytes are largely absent in the IL10KO HOM line (D). (E, F) Parasite-associated pathology measured as caecal lesion scores 6 dpi. (G, H) Body weight gain (BWG) over 6 days from time of challenge to sampling. All group sizes: n=10. Statistical significance calculated using one-way ANOVA with Tukey’s multiple comparison tests (A, B, G, H) or Kruskal-Wallis with Dunn’s multiple comparison tests (E, F); *p<0.05, ***p<0.001, ****p<0.0001, ns: not significant.
Figure 8 with 1 supplement
E. tenella infection time course in IL10 knockout (IL10KO) wild-type (WT) and homozygous (HOM) chickens.
(A) Parasite replication calculated as parasite genomes per host genome detected by quantitative PCR in genomic DNA extracted from caecal tissue. (B) Oocysts per gram (OPG) litter. (C) Parasite-associated pathology measured as caecal lesion scores. (D) Body weight gain (BWG) over 10 days from time of challenge to final sampling. (E) Heat map of differentially transcribed genes in the IL10KO HOM caeca relative to IL10KO WT chickens 0, 4, 6, and 8 days post-infection (dpi). 48 chicks per group, eight culled from each group per timepoint. Statistical significance calculated using mixed-effects model (REML) with Tukey’s multiple comparison tests (A, D), two-way ANOVA with Šídák’s multiple comparison tests (B), or Mann-Whitney U tests (C); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. Blue arrows indicate the time of sampling in the comparative phenotyping study (Figure 7).
Figure 8—figure supplement 1
Differential gene expression in the caecum of IL10 knockout (IL10KO) wild-type (WT) and homozygous (HOM) chickens infected with E. tenella.
Changes in transcription were analysed at 0 (A), 4 (B), 6 (C), and 8 (D) days post-infection.
Additional files
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Supplementary file 1
Number of IL10 knockout (IL10KO) and IL10-Enhancer knockout (IL10EnKO) wild-type (WT), heterozygous (HET), and homozygous (HOM) chicks hatched in the National Avian Research Facility (NARF) specified pathogen-free (SPF) chicken facility in the first (G1) and second (G2) generations.
n.d.: not determined.
- https://cdn.elifesciences.org/articles/106252/elife-106252-supp1-v1.docx
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Supplementary file 2
Routine vaccination schedule in the National Avian Research Facility (NARF) conventional chicken facility.
- https://cdn.elifesciences.org/articles/106252/elife-106252-supp2-v1.docx
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Supplementary file 3
PCR primer sequences and expected fragment sizes.
- https://cdn.elifesciences.org/articles/106252/elife-106252-supp3-v1.docx
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Supplementary file 4
Guide RNA and ssODN sequences.
Note the three substituted nucleotides (red, lowercase) and AvrII restriction site (CCTAGG, underlined) in the ssODN sequence.
- https://cdn.elifesciences.org/articles/106252/elife-106252-supp4-v1.docx
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Supplementary file 5
National Avian Research Facility (NARF) specified pathogen-free (SPF) screening.
- https://cdn.elifesciences.org/articles/106252/elife-106252-supp5-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/106252/elife-106252-mdarchecklist1-v1.pdf
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Interleukin 10 controls the balance between tolerance, pathogen elimination, and immunopathology in birds
eLife 14:RP106252.
https://doi.org/10.7554/eLife.106252.3
