Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4)

Abstract

Bacterial lipopolysaccharide (LPS) provokes a vigorous, generalized proinflammatory state in the infected host. Genetic regulation of this response has been localized to the Lps locus on mouse chromosome 4, through study of the C3H/HeJ and C57BL/10ScCr inbred strains. Both C3H/HeJ and C57BL/10ScCr mice are homozygous for a mutant Lps allele (Lpsd/d) that confers hyporesponsiveness to LPS challenge, and therefore exhibit natural tolerance to its lethal effects. Genetic and physical mapping of 1,345 backcross progeny segregating this mutant phenotype confined Lps to a 0.9-cM interval spanning 1.7 Mb. Three transcription units were identified within the candidate interval, including Toll-like receptor 4 (Tlr4), part of a protein family with members that have been implicated in LPS-induced cell signaling. C3H/HeJ mice have a point mutation within the coding region of the Tlr4 gene, resulting in a nonconservative substitution of a highly conserved proline by histidine at codon 712, whereas C57BL/ 10ScCr mice exhibit a deletion of Tlr4. Identification of distinct mutations involving the same gene at the Lps locus in two different hyporesponsive inbred mouse strains strongly supports the hypothesis that altered Tlr4 function is responsible for endotoxin tolerance.

Figure 1

Schematic representation of the Lps candidate region. (A) Genetic map of the Lps interval. A genetic map of mouse chromosome 4 was generated from haplotype analysis of 1,345 backcross progeny segregating the Lps phenotype. The Lps locus was mapped between D4Nds9 and D4Mit178. Genetic distances are given to the left in cM. (B) Physical map of the Lps region. Solid bar, genomic DNA. The order of markers was established based on the YAC, BAC, and P1 contig. Unique clone addresses are indicated. Arrow, the candidate Lps region. There are six unresolved recombination events between the proximal marker D4Nds9 and D4Mcg6, and five unmapped crossovers between D4Mcg25 and D4Mcg5. The position of the four transcription units and the pseudogene identified within the candidate region is shown immediately below the cloned contig. The direction of the transcription is shown for clone 1d8.

Figure 2

Expression analysis of three transcription units identified within the Lps candidate region. Mouse multiple-tissue Northern blots of polyA⁺ RNA (Clontech) were hybridized with (A) Tlr4; (B) clone 3f3; and (C) clone 1d8. β-actin was used as control. Exposure times were all 24 h and reflect the relative abundance of each transcript. RNA size markers (in kb) are shown on the left side of each autoradiogram.

Figure 3

Predicted 835 amino acid sequence of the mouse Tlr4 gene. Amino acid positions are numbered from the start of the 2.5-kb ORF. The protein sequences of mouse Tlr4 (top) and human TLR4 (bottom; EMBL/GenBank /DDBJ accession no. U93091) are aligned. Amino acid identity is shown between the two sequences; a + represents similarity between the corresponding pair of amino acid residues. The predicted transmembrane domain is underlined. The position of the proline to histidine substitution at position 712 associated with LPS hyporesponsiveness is highlighted in bold. These sequence data are available from EMBL/ GenBank/DDBJ under accession no. AF110133.

Figure 4

Tlr4 mutation analysis in C3H/HeJ and C57BL/10ScCr. (A) C3H/HeJ Lps mutation. Sequence analysis of Tlr4 shows the missense mutation, a C to A transversion (Pro⁷¹²His). This change is present only in C3H/HeJ mice. A silent mutation (C to T substitution, Asn⁷¹⁹) is also present in several inbred strains of mice, including A/J and BALB/cJ. (B and C) C57BL/10ScCr Lps mutation. (B) RT-PCR of spleen mRNA from C57BL/6J (lanes 3, 6, 9, 12, 17, 20, 23, and 26), C3H/HeJ (lanes 4, 7, 10, 13, 18, 21, 24, and 27), and C57BL/10ScCr (lanes 5, 8, 11, 14, 19, 22, 25, and 28) using six different combinations of primers. Molecular size markers λ DNA-HindIII digest (lanes 1 and 15) and φX-174 RF HincII digest (lanes 2 and 16) are shown. Primer positions are given in brackets according to the position of the first coding ATG. Lanes 3–5: fragment A (696 bp) was amplified with primers 5′-TCTGCGCTGCCACCAGTTAC-3′ (−69) and 5′-ACATGTCTAAAGAGAGATTGAC-3′ (627). Lanes 6–8: fragment B (1,200 bp) was amplified with primers 5′-GCTGGATTTATCCAGGTG-3′ (242) and 5′-GAAAGAATTGCCAGCCATTTT-3′ (1,442). Lanes 9 and 10: fragment C (2,700 bp) was amplified with primers 5′-GCTGGATTTATCCAGGTG-3′ (242) and 5′-AAATTGGAATGAAGACCTCTCA-3′ (2,942). Lanes 17–19: fragment D (409 bp) was amplified with primers 5′-TGACACCCTCCATAGACTTC-3′ (1,541) and 5′-GGTATATCAGAAATGCTACA-3′ (1,950). Lanes 20–22: fragment E (641 bp) was amplified with primers 5′-GTCCTTGAGAAGGTTGAGAAGTCCC-3′ (2,301) and 5′-AAATTGGAATGAAGACCTCTCA-3′ (2,942). Lanes 23–25: fragment F (299 bp) was amplified with primers 5′-GAAGGAAGTAGCACTGACACCTTC-3′ (2,643) and 5′-AAATTGGAATGAAGACCTCTCA-3′ (2,942). Lanes 12–14 and 26–28: Gapdh (459 bp) was amplified with primers 5′-TTTGTGATGGGTGTGAACCACGAG-3′ and 5′-GGAGACAACCTGGTCCTCAGTGTA-3′. A single Tlr4 product of the expected size is seen only in RT-PCR of spleen mRNA from C57BL/6J and C3H/HeJ, although Gapdh mRNA was amplified from all RT-PCR samples including C57BL/10ScCr. (C) Southern blot analysis of chromosomal rearrangement associated with the C57BL/10ScCr Lps allele. Southern blot of a Tlr4 partial cDNA (position 242–1442) hybridized to C57BL/6J (lanes 1 and 5), C57BL/10ScCr (lanes 2 and 6), C3H/HeJ (lanes 3 and 7), and DBA/2J (lanes 4 and 8) DNA digested with BamHI (1– 4) or HindIII (–8). Size markers (in kb) are indicated to the left of the autoradiogram.

Figure 4

Tlr4 mutation analysis in C3H/HeJ and C57BL/10ScCr. (A) C3H/HeJ Lps mutation. Sequence analysis of Tlr4 shows the missense mutation, a C to A transversion (Pro⁷¹²His). This change is present only in C3H/HeJ mice. A silent mutation (C to T substitution, Asn⁷¹⁹) is also present in several inbred strains of mice, including A/J and BALB/cJ. (B and C) C57BL/10ScCr Lps mutation. (B) RT-PCR of spleen mRNA from C57BL/6J (lanes 3, 6, 9, 12, 17, 20, 23, and 26), C3H/HeJ (lanes 4, 7, 10, 13, 18, 21, 24, and 27), and C57BL/10ScCr (lanes 5, 8, 11, 14, 19, 22, 25, and 28) using six different combinations of primers. Molecular size markers λ DNA-HindIII digest (lanes 1 and 15) and φX-174 RF HincII digest (lanes 2 and 16) are shown. Primer positions are given in brackets according to the position of the first coding ATG. Lanes 3–5: fragment A (696 bp) was amplified with primers 5′-TCTGCGCTGCCACCAGTTAC-3′ (−69) and 5′-ACATGTCTAAAGAGAGATTGAC-3′ (627). Lanes 6–8: fragment B (1,200 bp) was amplified with primers 5′-GCTGGATTTATCCAGGTG-3′ (242) and 5′-GAAAGAATTGCCAGCCATTTT-3′ (1,442). Lanes 9 and 10: fragment C (2,700 bp) was amplified with primers 5′-GCTGGATTTATCCAGGTG-3′ (242) and 5′-AAATTGGAATGAAGACCTCTCA-3′ (2,942). Lanes 17–19: fragment D (409 bp) was amplified with primers 5′-TGACACCCTCCATAGACTTC-3′ (1,541) and 5′-GGTATATCAGAAATGCTACA-3′ (1,950). Lanes 20–22: fragment E (641 bp) was amplified with primers 5′-GTCCTTGAGAAGGTTGAGAAGTCCC-3′ (2,301) and 5′-AAATTGGAATGAAGACCTCTCA-3′ (2,942). Lanes 23–25: fragment F (299 bp) was amplified with primers 5′-GAAGGAAGTAGCACTGACACCTTC-3′ (2,643) and 5′-AAATTGGAATGAAGACCTCTCA-3′ (2,942). Lanes 12–14 and 26–28: Gapdh (459 bp) was amplified with primers 5′-TTTGTGATGGGTGTGAACCACGAG-3′ and 5′-GGAGACAACCTGGTCCTCAGTGTA-3′. A single Tlr4 product of the expected size is seen only in RT-PCR of spleen mRNA from C57BL/6J and C3H/HeJ, although Gapdh mRNA was amplified from all RT-PCR samples including C57BL/10ScCr. (C) Southern blot analysis of chromosomal rearrangement associated with the C57BL/10ScCr Lps allele. Southern blot of a Tlr4 partial cDNA (position 242–1442) hybridized to C57BL/6J (lanes 1 and 5), C57BL/10ScCr (lanes 2 and 6), C3H/HeJ (lanes 3 and 7), and DBA/2J (lanes 4 and 8) DNA digested with BamHI (1– 4) or HindIII (–8). Size markers (in kb) are indicated to the left of the autoradiogram.