IL10KO and IL10EnKO editing strategies.

A: Schematic of the pipeline used to create gene-edited chickens using embryonic PGCs and the iCaspase9 sterile surrogate host line. Male PGCs carrying the desired biallelic (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 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 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.

Confirmation of the IL10 mutation in bone marrow-derived macrophages.

A: IL10 protein levels were measured by capture ELISA for LPS-stimulated BMDMs derived from Day 18 IL10KO WT (n=4), HET (n=2), HOM (n=4) and 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 negligeable (<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 neutralizing anti-IL10 antibody ROS-AV163. 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 IL10KO HOM BMDMs; nitrite levels were also increased in IL10KO HET and IL10EnKO HOM BMDMs. Addition of neutralizing anti-IL10 antibody did not significantly affect nitrite levels. 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.

Growth curves and histopathological analyses of the gastrointestinal tract of IL10-deficient chickens in SPF and conventional facilities.

A: Growth curves for IL10KO WT (n=3 to 8), HET (n=11 to 16) and HOM (n=2 to 4) hens raised in the 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 IL10EnKO WT (n=2 to 4), HET (n=7 to 11) and HOM (n=5 to 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 to 10) and HOM (n=5 to 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 to 3. Data displayed as mean with SD (A, B and D) or as median with 95% confidence interval (C, E and 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.

Net replication of C. jejuni and S. Typhimurium in the caeca of IL10KO WT, HET and 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 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.

Eimeria tenella replication and impact on host pathology and performance in WT, HET and HOM G2 populations of IL10KO and IL10EnKO chickens.

A-B: Parasite replication calculated as parasite genomes per host genome detected by quantitative PCR in genomic DNA extracted from caecal tissue six days post-infection (dpi). C-D: Haematoxylin and eosin stained caecal tissues collected from IL10KO WT and HOM chickens six days post Eimeria 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 six dpi. G-H: Bodyweight gain (BWG) over six 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.

Eimeria tenella infection time course in IL10KO WT and 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: Bodyweight gain (BWG) over ten 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 5).