Experiment
Previous studies revealed a major, differential contribution of Type I IFN to the development of Lyme arthritis in C3H mice, which could be overcome by blocking the Type I IFN receptor signaling with neutralizing mAb or by gene ablation (19, 20). It was noted that Bbaa1, a QTL controlling the severity of Lyme arthritis on Chr4 encompassed a cluster of 15 Type I IFN genes, prompting a more mechanistic analysis of the role of Bbaa1 as a regulator of Lyme arthritis severity (30, 32).
In the current study the analysis of QTL Bbaa1 on Chromosome 4 was extended and the type I IFN gene cluster was identified as positional candidates for Bbaa1. Two mouse strains, C57BL/6 and C3H/He, display extremes of arthritis severity and have been used extensively as experimental models for study of disease. Reciprocal interval specific congenic lines (ISCL) were developed between C3H/HeNCrl (C3H) and C57BL/6Ncr (B6) mice to assess the contribution of Bbaa1 to Lyme arthritis and RA, and the dependence on production of type I IFN.
Reciprocal ISCL for Bbaa1 on Chr 4 were generated and indicated as B6.C3-Bbaa1(9.32-94.96) Mbp and C3.B6-Bbaa1(3.58-150.8) Mbp. B6, C3H, B6.C3-Bbaa1 and C3.B6-Bbaa1 mice were infected with 2 x 104 B. burgdorferi (strain N40) and arthritis was assessed at 4 weeks post infection. Ankle measurements were obtained using a metric caliper before and at 4 weeks of infection. IFNAR1 blocking mAb MAR1-5A3 or isotope control (Bio-XCell) was administered by a single injection the day before infection with B. burgdorferi or the administration of K/BxN serum. (K/BxN serum was collected from KRN/NOD offspring at the peak of spontaneous arthritis (9 weeks)). K/BxN serum was administered by injection on days 0 and 2. Gene expression of joint tissue was examined in the tibiotarsal joints. Bone marrow-derived macrophages (BMDMs) were isolated from the femurs and tibias. Statistical analysis was performed using Prism 5.0c software. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
Experimental results demonstrated that Bbaa1 regulates arthritis severity on the B6 background, [Fig 1]. More severe ankle swelling and arthritic lesions were seen in the infected B6.C3-Bbaa1 mice than in the parental B6 mice. Although the B6.C3-Bbaa1 congenic interval was large (9.32-94.97 Mbp) and encoded numerous genes, it nevertheless encompassed the IFN/cluster (88.5-88.7 Mbp) that encode Type I IFNs implicated in Lyme arthritis. All IFN and IFN proteins signal through the IFNAR receptor, comprised of IFNAR1 and IFNAR2 chains.
To determine if the greater arthritis in B6.C3-Bbaa1 mice relative to B6 mice was dependent on Type I IFN, B6.C3-Bbaa1 mice were treated with a blocking mAb to IFNAR1 component of the Type I IFN receptor (MAR1-5A3) one day prior to infection. B6.C3-Bbaa1 mice treated with isotype control mAb developed more severe Lyme arthritis than B6 mice as assessed by ankle measurement and lesion scoring [Fig. 2], similar to the results of Fig. 1. Administration of the receptor blocking mAb reduced arthritis severity to levels indistinguishable from wild type B6. Thus, the increased arthritis severity of B6.C3- Bbaa1 mice was a function of Type I IFN responses dependent on feed forward amplification through the IFNAR1 receptor, and identified the IFN cluster as a positional candidate for Bbaa1.
Type I IFNs have also been identified in patients with various manifestations of Lyme disease, including skin lesions and cognitive deficits, and have been implicated in in B lymphocyte accumulation in the lymph nodes of infected mice.
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