Figure 1.
Identification and mapping of the Ity16 pedigree.
A) Breeding scheme used to identify and map the mutant family. Females and males are represented by circles and squares respectively with black representing the homozygous mutant alleles, gray representing the heterozygous alleles, and white representing the wild type alleles. B) Survival curve of the G3 mice from the Ity16 pedigree is represented by the solid line (n = 30) while wild type 129S1 (n = 5) mice are represented by dotted lines. Log-Rank (Mantel-Cox) p = 0.01. C) Linkage analysis in 8 Salmonella-susceptible and 14 Salmonella-resistant mice identifies a significant peak on chromosome 8. D) Fine mapping of the Ity16 locus to a 2.5 Mb region on chromosome 8. Black fill represents homozygous 129S1 allele. White fill represents heterozygous or homozygous DBA/2 genotypes. E) Survival curves of Ity16 mice according to their genotypes at peak marker on chromosome 8. Solid line represents homozygous 129S1 alleles (n = 6), dashed line represents heterozygous (n = 12), and dotted line represents homozygous DBA/2 alleles (n = 12). Log-Rank (Mantel-Cox) for Ank1+/+ and Ank1Ity16/Ity16 p<0.0001, Ank1+/+ and Ank1+/Ity16 p = 0.001.
Figure 2.
Ity16 mutant mice present massive splenomegaly, moderate increase in kidneys and heart weights and bilirubinemia.
A) Body weight of wild type littermates and Ity16 mice by sex (males are represented by squares, females by circles) at 7 weeks (open and black fill) and 24 weeks (open and black fill) n = 3 per sex and genotype. B) Serum bilirubin levels were measured at day 0 and day 2 post infection in wild type (open circle), heterozygous (gray circle) and Ity16 mutant (black circle) mice. C) Increased spleen, kidney and heart weights in Ity16 mutant (black fill) mice compared to wild type littermates (open fill). The data are presented by sex (males are squares, females are circles). Results are representative of at least two experiments. An * represents a P-value of less than 0.05; ** represents a P-value of less than 0.001; *** represents a P-value of less than 0.0001.
Table 1.
Hematologic parameters in Ity16 mutant, heterozygous and wild type mice before (day 0) and after (day 2) infection with Salmonella Typhimurium.
Figure 3.
Ity16 mutant mice carry mutation within the Ank1 gene.
A) A C to T transition (arrow) at cDNA position 4069 (c.4069C>T) resulting in a stop codon at amino acid position 1357 (p.Gln1357Ter) was detected in Ank1Ity16/Ity16 mutant. The Ity16 mutation is located in exon 33 using the exon numbering of the Ank1 transcript ID ENSMUST00000121802 (Ensembl build 37). B) Schematic representation of the different domains of ANK1 (adapted from [57]) and location of mutations previously identified in human (small ticks) [18] and mouse ANK1 [13], [14], [15], [16], [17]. The Ity16 allele is shown together with two spontaneous ANK1 recessive mutations (nb and pale) and three dominant mutants (RBC2, M1Wlst and MRI23420). C) Immunoblot of RBC ghosts prepared from wild-type (+/+), heterozygous (+/m) and mutant Ank1 (m/m) littermates probes with ANK1 antibody. The size of the molecular markers in kDa are shown on the left. An expected band of about 210 kDa was observed in samples from the wild-type and heterozygous mice while this band or any smaller bands corresponding to a truncated version of the protein could not be detected in mutant mice.
Figure 4.
Bacterial load in spleen, liver and kidney after infection with Salmonella Typhimurium in Ity16 mutant, heterozygous and wild type mice.
The bacterial load was measured 2 days after infection in the spleen (A), liver (B), and kidney (C) of wild type aged 7 weeks (open circle), and 24 weeks (open circle), heterozygous mice at 7 weeks (gray circle) and 24 weeks (gray circle) and Ity16 mutant mice at 7 weeks (filled circle) and 24 weeks (filled circle) (n = 3–5 per genotype and age). Ity16 mutant mice present higher bacterial load in the liver and kidneys compared to heterozygous and wild type littermates. Bacterial load of Salmonella Typhimurium and ΔtonB Salmonella Typhimurium was measured 2 days after infection in spleen (D) and liver (E) of wild type (open circle), heterozygous (gray circle) and Ity16 (black circle) mice aged 7–12 weeks (n = 3–8 per genotype). Results are representative of at least two experiments. An * represents a P-value of less 0.05; **represents a P-value of less than 0.001; ***represents a P-value of less than 0.0001.
Figure 5.
Ity16 mutant mice present elevated blood urea nitrogen (BUN), alanine transaminase (ALT) and asparatate transaminase (AST) during Salmonella infection.
BUN (A), ALT (B) and AST (B) levels were measured at day 0 (prior to infection) and day 2 post infection in 7 week old Ank1+/+ wild type, Ank1+/Ity16 heterozygous and Ank1Ity16/Ity16 mutant mice. The values for each individual mouse are shown (n = 3–8 per genotype). Wild type mice are represented by open circles, heterozygous by gray circles and Ity16 mutant by black circles. The high levels of BUN levels detected in mutant mice at day 0 most probably reflect the nephrotoxicity of iron accumulation in the kidney. During infection BUN, ALT and AST levels increased in mutant mice denoting kidney and liver damage. An *represents a P-value of less than 0.05; *** represents a P-value of less than 0.0001.
Figure 6.
Tissue expression of genes involved in iron metabolism in 7 week old Ank1+/+ wild type and Ank1Ity16/Ity16 mutant mice.
Real-time PCR expression of liver Hamp (A), kidney Epo (B), spleen, liver and kidney Slc40a1 (C), and spleen and liver Gdf15 (D). The relative mRNA levels at day 0 and day 2 post infection are shown in Ank1+/+ wild type (clear bar, n = 3) and Ank1Ity16/Ity16 mutant (black bar, n = 3) mice. Epo mRNA levels are represented on a LOG10 scale. (E) Survival curves of Hamp knock out mice (129S6.B6*129S2-Hamptm1Svl) infected with 8000 CFUs of Salmonella Typhimurium. Solid line represents homozygous Hamp−/− knock out (n = 8), dashed line represents heterozygous Hamp+/− (n = 12), and dotted line represents homozygous wildtype Hamp+/+ alleles (n = 10). Log-Rank (Mantel-Cox) for Hamp+/+ and Hamp−/− p = 0.0071. An *represents a P-value of less than 0.05; ** represents a P-value of less than 0.001; *** represents a P-value of less than 0.0001.
Figure 7.
Cytokine profiles in 7 week-old Ank1+/+ wild type and Ank1Ity16/Ity16 mutant mice during infection with Salmonella Typhimurium.
Relative spleen, liver and kidney mRNA levels are shown for Il1 (A),Il6 (B), and Hmox1 (C) at day 0 and day 2 post infection. Ank1+/+ wild type mice (n = 3) are represented by clear bar and Ank1Ity16/Ity16 mutant mice (n = 3) by a black bar. All values are compared to wild type mRNA levels at day 0 and presented on a LOG10 scale. An * represents a P-value of less than 0.05; ** represents a P-value of less than 0.001; *** represents a P-value of less than 0.0001.
Figure 8.
Characterization of Ank1+/Ity16 heterozygous mice.
RBC counts (A) and bacterial burden in spleen, liver and kidney (D) were measured in 7 week old Ank1+/+ wild type and Ank1+/Ity16 heterozygous mice at day 0 (prior infection), day 2 and day 6 post infection. Wild type Ank1+/+ mice are represented by clear circles and Ank1+/Ity16 heterozygous mice by gray circles. Relative kidney mRNA levels are shown for Hamp (C), Il1 (E), IL6 (F) and Epo (G) at day 0, day 2 and day 6 post infection. (B) Relative liver Hamp levels at day 0, day 2 and day 6 post infection. Wild type Ank1+/+ mice are represented by clear bars (n = 3 for day 0 and day 2; n = 4 for day 6) and Ank1+/Ity16 heterozygous mice by gray bars (n = 3 for day 0 and day 2; n = 4 for day 6). (H) Survival curves of Ank1+/+ wild type (n = 5; open circle) and Ank1+/Ity16 heterozygous (n = 5; gray circle) mice with (n = 5; dashed or dotted line) or without (n = 5; solid line) HAMP treatment infected with 5000 CFUs of Salmonella Typhimurium. Log-Rank (Mantel-Cox) for Ank1+/Ity16 heterozygous with and without HAMP treatment p = 0.008. The experiment was repeated twice. An * represents a P-value of less than 0.05; ** represents a P-value of less than 0.001; *** represents a P-value of less than 0.0001.