Experiment
Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most prevalent arthropod-borne illness in the United States. Inbred strains of laboratory mice exhibit consistent differences in arthritis severity following infection with B. burgdorferi, with the C3H/He or C3H/HeNCr (C3) mouse displaying the most severe disease and the C57BL/6 (B6) mouse displaying less severe disease. The focus of the current study was the identification of the genes responsible for disease severity following infection.
Previously, intercross populations between C3H/HeNCr and C57BL/6NCr (or BALB/c) mice led to the identification of multiple B. burgdorferi arthritis associated (Bbaa) quantitative trait loci (QTL) on 5 chromosomes [J:52017]. Four individual crosses identified Bbaa2 on mouse chromosome 5, exhibiting the strongest linkage to disease severity, with a maximum lod score of 10.2 [J:73060]. Subsequently a B6.C3-Bbaa2C3H/HeNCrBbaa3C3H/HeNCr/Wjj congenic mouse line was generated, where B6.C3-Bbaa2Bbaa3 from susceptible C3 mice was isolated on an otherwise uniform resistant B6 genetic background, and found that the congenic mice exhibited increased Lyme arthritis severity, with joint inflammation and histopathology closely resembling the human disease [J:151329].
In the current study, through additional backcrossing to the parental B6 line, 15 advanced B6.C3-Bbaa2C3H/HeNCr/Wjj congenic mouse lines harboring subintervals of Bbaa2C3H/HeNCr/Wjj from 120.3 to 141.2 Mbp were developed [Mbp, according to the NCBI37/mm9 Mouse Genome Assembly]. After infection with B. burgdorferi, the various subinterval congenic lines exhibited a wide spectrum of disease severity, as assessed quantitatively by ankle swelling measurements. Compared with B6, congenic mice harboring C3-derived intervals from 129.0-130.5 Mbp (P < 0.01), 133.5-141.2 Mbp (P < 0.05), and 125.3-128.2 Mbp (P <0.05) within Bbaa2 exhibited significantly more severe disease. The ankle swelling data also supported the presence of a negative regulatory element within Bbaa2. The 129.0-130.5 Mbp and 133.5-141.2 Mbp intervals, increased in the categorical traits of pathology score and neutrophil (PMN) infiltration cosegregated with ankle swelling. Consequently, the intervals have been designated Bbaa2a and Bbaa2b, respectively.
The Sanger SNP resequencing database indicated that the Bbaa2a interval harbored only 1 high-confidence coding nonsynonymous GA polymorphism differing between B6 and C3H strains, which caused a T87I amino acid change in the ubiquitously expressed lysosomal enzyme Gusb. The C3H strain is known to carry a functionally hypomorphic Gusbh allele, which confers a 70%-90% reduction in enzymatic activity in the serum and various tissues. The Sanger SNP database indicated that only 3 of the 18 included strains, C3H/HeJ, AKR/J, and CBA/J, share this Gusb coding variant. Both C3H and AKR/J have previously been shown to develop severe Lyme arthritis.
The availability of a spontaneous Gusb mutant mouse line on the resistant B6 genetic background, B6.C3-Gusbmps-2J/BrkJ (GusbNull), allowed the determination of the impact of GUSB loss of function in a second, independent mouse line. GusbNull mice exhibit no defect in host defense (Figure 2A) despite expressing only 1% of normal GUSB levels in homozygotes (Figure 2B). Infected homozygous GusbNull mice developed maximally severe Lyme arthritis, while heterozygous littermates carrying 1 functional Gusbb allele were protected.
To clarify which pool of GUSB is responsible for its effect on arthritis, radiation chimeras were generated in all pairwise combinations between B6 and B6.C3-Bbaa2 congenic mice. The results indicated that GUSB hypomorphism primarily modulates disease severity within joint-resident, radiation-resistant cells and that serum GUSB levels are not determinative. This suggests that GUSB hypomorphism acts through a localized, cell-intrinsic mechanism to initiate the development of inflammatory arthritis.
To determine the magnitude of the Gusb effect, a transgene driving ubiquitous mouse Gusbb expression (Figure 4A) was used to produce C3H/HeN-CAG-Gusbb transgenic mice, GusTg. Following infection with B. burgdorferi, GusbTg progeny exhibited a profound and highly significant (P < 0.001) reduction in disease severity (Figure 4C) relative to WT C3H control mice. The findings argue that among the many Bbaa loci previously identified to regulate Lyme arthritis severity in C3H mice, Gusb is a key regulator.
Authors suggest that the identification of Gusb as a key regulator of murine Lyme and rheumatoid arthritis severity provides a sound scientific basis for future investigations into serum GUSB or GAG levels as potential biomarkers of human susceptibility to developing chronic or severe inflammatory arthritis.
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