Experiment
The observation that Borrelia burgdorferi-induced arthritis is severe in C3H/HeNCr (C3) mice and milder in C57BL/6NCr (B6) mice has allowed a forward genetics approach for the identification of genetic elements that regulate the arthritis response. Quantitative trait loci (QTL) on five chromosomes (Chr) were identified previously in segregating crosses between C3H and B6 mice and collectively designated B. burgdorferi arthritis-associated (Bbaa) QTL.
To evaluate the six previously identified Bbaa QTL with the broadest arrays of arthritis-associated phenotypes exhibiting linkage in multiple analyses, and as the first step toward positionally cloning the respective genes, reciprocal interval-specific congenic lines (ISCL) were generated encompassing Bbaa1 (Chr 4), Bbaa2-Bbaa3 (Chr 5), Bbaa4 (Chr 11), Bbaa6 (Chr 12), and Bbaa12 (Chr 1). Of the five Bbaa QTL studied, Bbaa2-Bbaa3, Bbaa4, and Bbaa6 were associated with significant differences in disease severity between the congenic and the parental background strains.
C3.B6-Bbaa2C57BL/6NCrBbaa3C57BL/NCr/Wjj (Chr 5, 72.29-137.53) and B6.C3-Bbaa2C3H/HeNCrBbaa3C3H/HeNCr/Wjj (Chr 5, 72.29-141.16) congenic mouse lines were generated by the introgression of the region of Chr 5 spanning both Bbaa2 and Bbaa3 from C3H/HeNCr or C57BL/6NCr mice into the reciprocal parental strain. Similarly, C3.B6-Bbaa1C56BL/6NCr/Wjj (Chr 4, Mbp 3.58-150.08), B6.C3-Bbaa1C3H/HeNCr/Wjj (Chr 4, Mbp 9.32-94.07), C3.B6-Bbaa4C57BL/6NCr/Wjj (Chr 11, Mbp 25.86-103.24), B6.C3-Bbaa4C3H/HeNCr/Wjj (Chr 11, Mbp 17.18-101.75), C3.B6-Bbaa6C57BL/6NCr/Wjj (Chr12, Mbp 73.39-116.11), B6.C3-Bbaa6C3H/HeNCr/Wjj (Chr12, Mbp 82.01-111.54), C3.B6-Bbaa12C57BL/6NCr/Wjj (Chr 1,Mbp 116.02-96.25), and B6.C3-Bbaa12C3H/HeNCr (Chr1, Mbp 171.14-196.25) were generated by marker-assisted selection. (Mouse genome build not specified).
Mice between 5 and 6 weeks of age were infected by intradermal injection with 2 x 103 bacteria of B. burgdorferi N40 isolate. Rear ankle joints were measured at the time of infection and at 4 weeks after infection by using a metric caliper. The joint sections were evaluated blindly and scored for the severity of injury according to a subjective scale ranging from 0 to 5. A score of 0 indicated no lesions, and scores of 1, 2, 3, 4, and 5 indicated minimal, mild, moderate, marked, and severe lesions, respectively . The overall lesion score represented a combined assessment of neutrophil infiltration, mononuclear cell infiltration, tendon sheath thickness, and reactive-reparative responses.
Total RNA from rear ankle tissues was isolated by acid guanidine extraction. Gene expression analyses of B. burgdorferi-infected mice were performed using equal amounts of total RNA from the ankle tissues of five to eight individual mice of each genotype. Transcripts found to have a change in levels in infected tissues of twofold or greater relative to the levels in uninfected tissues were considered to be differentially expressed, whereas other transcripts were considered to be unchanged.
Statistical analyses were performed using GraphPad InStat3 version 3.0b for Macintosh (GraphPad Software, San Diego, CA; http://www.graphpad.com). Values of P of < 0.05 were considered significant.
Results (Table 1, Fig 2) indicated that compared to B6 mice, B6.C3-Bbaa2-Bbaa3 mice displayed significantly increased arthritis severity at 4 weeks following B. burgdorferi infection; both ankle swelling and histopathology increased relative to B6. C3.B6-Bbaa2-Bbaa3 mice had significantly less severe arthritis than infected C3 mice; both ankle swelling and histopathology decreased relative to C3 mice. Both Bbaa2-Bbaa3 congenics had intermediate degrees of arthritis severity relative to those seen in the parental strains.
B6.C3-Bbaa4 mice infected with B. burgdorferi developed significantly more severe arthritis than did B6 mice; both ankle swelling and histopathology increased relative to B6. Ankle tissue samples mounted onto slides were scored blindly and also revealed increased arthritis severity for the categories of tendon sheath thickness, polymorphonuclear leukocyte (PMN) infiltration, reactive-reparative responses, and overall lesion severity. In the reciprocal congenic, C3.B6-Bbaa4, the degree of arthritis severity at 4 weeks post infection was significantly lower than that seen in C3H mice. Similarly, multiple measures of arthritis severity, including ankle swelling, sheath thickness, PMN infiltration, reactive-reparative responses, and the over-all lesion score, were significantly reduced.
C3.B6-Bbaa6 mice also displayed significantly reduced arthritis severity compared with that in C3H mice. However, arthritis severity was not significantly increased in B6.C3-Bbaa6 mice relative to that in wild-type B6 mice. B6.C3-Bbaa1, B6.C3- Bbaa12, and C3.B6-Bbaa12, all failed to display a consistent, significant, penetrant phenotype.
Ninety-one genes identified previously (using the GeneChip 2.0A array) as being induced more than twofold compared to those in samples from uninfected mice were also identified in the present study (using the GeneChip 2.0 array) as being upregulated more than two- fold. Only two of the robustly upregulated transcripts, those for Cxcl9 and Cxcl10, were both found within Bbaa2-Bbaa3, were encoded by penetrant QTL. Gene expression profiling of joint tissues from infected congenic mice or their BMMs indicated that a previously identified IFN-induced profile is not regulated by Bbaa2-Bbaa3, Bbaa4 or Bbaa6 in joints or macrophages.
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