Figure 1.Cruyff mice exhibited reduced TNFα secretion in response to the TLR4 ligand, LPS. TNFα levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 2.Cruyff mice exhibited resistance to macrophage necroptosis in response to the TLR4 ligand, LPS. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 3.Cruyffmice secreted decreased amounts of IL-1β in response to priming with lipopolysaccharide (LPS) followed by nigericin treatment. IL-1β levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
The cruyff phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4213, some of which showed reduced TNFα secretion from macrophages in response to the Toll-like receptor 4 (TLR4) ligand, lipolysaccharide (LPS) (Figure 1) and resistance to LPS-induced macrophage necroptosis (Figure 2). Some mice also exhibited attenuated inflammatory responses related to decreased secretion of the proinflammatory cytokine interleukin (IL)-1β in response to priming with lipopolysaccharide (LPS) followed by nigericin treatment (Figure 3).
Nature of Mutation
Figure 4.Linkage mapping of the increased LPS-induced necroptosis of macrophage cells using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 37 mutations identified in the G1 male of pedigree R4213 (X-axis). Normalized phenotype data are shown for single locus linkage analysis with consideration of G2 dam identity. Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.
Whole exome HiSeq sequencing of the G1 grandsire identified 37 mutations. All of the above phenotypes were linked by continuous variable mapping to a mutation in Tlr4: a T to A transversion at base pair 66,840,326 (v38) on chromosome 4, or base pair 12,516 in the GenBank genomic region NC_000070. The strongest association was found with a recessive model of linkage to the resistance to LPS-induced macrophage necroptosis, wherein 9 variant homozygotes departed phenotypically from 4 homozygous reference mice and 12 heterozygous mice with a P value of 1.859 x 10-19 (Figure 4).
The mutation corresponds to residue 1,636 in the mRNA sequence NM_021297 within exon 3 of 3 total exons.
The mutated nucleotide is indicated in red. The mutation results in an isoleucine (I) to asparagine (N) substitution at position 452 (I452N) in the TLR4 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 1.000).
Figure 4. Protein and domain structure of TLR4. A) Schematic representation of TLR9 based on crystalized structures of mouse TLR3 LRR (PBD 3CIG) and human TLR2 TIR (1FYW) domains. The residue affected by the Lps3 mutation is highlighted. 3D image was created using UCSF Chimera. B) TLR4 is an 835 amino acid protein with an extracellur domain (pink) of leucine rich repeats (LRR), a short transmembrane domain and a cytoplasmic Toll/Interleukin-1 receptor (TIR) domain. The cruyff mutation (red asterisk) results in an esults in substitution of isoleucine (I) 425 to an asparagine (I452N) in the TLR4 protein. This image is interactive. Click on the image to view other mutations found in TLR4 (red). Click on the mutations for more specific information.
TLR4 is a type I integral membrane glycoprotein containing 835 amino acids. TLR4 has 22 predicted leucine-rich repeats (LRRs) in its ectodomain at the N-terminal half of the protein (1-3), a transmembrane domain, and a cytoplasmic Toll/IL-1R (TIR) domain (Figure 5). The cruyff mutation results in substitution of isoleucine 452 to an asparagine (I452N); amino acid 452 is located in LRR16.
Please see the record for lps3 for information about Tlr4.
TLR4 is the receptor for LPS (4). Stimulation of TLR4 by LPS activates two branches of signaling, one defined by early NF-κB activation (MyD88-dependent pathway, mediated by MyD88), and another distinguished by late NF-κB activation as well as interferon responsive factor (IRF)-3 activation leading to type I IFN production and costimulatory molecule upregulation (MyD88-independent pathway, mediated by Trif) (5-7). The MyD88-dependent pathway activates expression of target genes including interleukin (IL)-6, IL-1, TNF, IL-12p40 and type I interferon (IFN), cytokines required for the inflammatory response. The MyD88-independent pathway results in the production of type I IFN. The reduction in TLR4-associated responses in cruyff indicates that the mutation results in loss of TLR4 function.
cruyff(F):5'- GTAGAAATGCACTGAGCTTTAGTGG -3'
cruyff(R):5'- CCCCAAGATATTTGTTCCAATTGAC -3'
cruyff_seq(F):5'- GCTGTTCTTATTCTGATTTGGGAAC -3'
cruyff_seq(R):5'- CCAATTGACATTTAGAAAGATCCAGG -3'