Figure 1.Guardiola 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.Guardiolamice secreted decreased amounts of IL-1β in response to priming with lipopolysaccharide (LPS) followed by flagellin 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 Guardiola phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4417, some of which showed reduced TNFα secretion from macrophages in response to the Toll-like receptor 4 (TLR4) ligand, lipolysaccharide (LPS) (Figure 1) and attenuated inflammatory responses related to decreased secretion of the proinflammatory cytokine interleukin (IL)-1β in response to priming with lipopolysaccharide (LPS) followed by flagellin treatment (Figure 2).
Nature of Mutation
Figure 3.Linkage mapping of the decreased TNFα secretion in response to LPS using an additive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 50 mutations identified in the G1 male of pedigree R4417 (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 50 mutations. Both of the above anomalies were linked by continuous variable mapping to a mutation in Tlr4: an A to T transversion at base pair 66,839,303 (v38) on chromosome 4, or base pair 11,493 in the GenBank genomic region NC_000070 for the Tlr4 gene. The strongest association was found with an additive model of linkage to the normalized response to LPS, wherein three variant homozygotes and 10 heterozygotes departed phenotypically from four homozygous reference mice with a P value of 8.88 x 10-8 (Figure 3).
The mutation corresponds to residue 613 in the mRNA sequence NM_021297.2 within exon 3 of 3 total exons.
The mutated nucleotide is indicated in red. The mutation results in an asparagine (N) to isoleucine (I) substitution at position 111 (N111I) in the TLR4 protein.
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 Guardiola mutation (red asterisk) results in substitution of asparagine 111 to an isoleucine (N111I) 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 4). The Guardiola mutation results in substitution of asparagine 111 to an isoleucine (N111I); amino acid 111 is located in LRR3.
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 Guardiola indicates that the mutation results in loss of TLR4 function.
Guardiola(F):5'- AATCTGCAGAGTTCCTCTCCTG -3'
Guardiola(R):5'- CCACATGTACTAGGTTCGTCAG -3'
Guardiola_seq(F):5'- ATCACCTGTTTTGCTCTGTACAG -3'
Guardiola_seq(R):5'- CACATGTACTAGGTTCGTCAGATTGG -3'