Borrelia burgdorferi arthritis-associated locus Bbaa1 regulates Lyme arthritis and K/B×N serum transfer arthritis through intrinsic control of type I IFN production

Abstract

Localized upregulation of type I IFN was previously implicated in development of Borrelia burgdorferi-induced arthritis in C3H mice, and was remarkable due to its absence in the mildly arthritic C57BL/6 (B6) mice. Independently, forward genetics analysis identified a quantitative trait locus on Chr4, termed B. burgdorferi-associated locus 1 (Bbaa1), that regulates Lyme arthritis severity and includes the 15 type I IFN genes. Involvement of Bbaa1 in arthritis development was confirmed in B6 mice congenic for the C3H allele of Bbaa1 (B6.C3-Bbaa1), which developed more severe Lyme arthritis and K/B×N model of rheumatoid arthritis (RA) than did parental B6 mice. Administration of a type I IFN receptor blocking mAb reduced the severity of both Lyme arthritis and RA in B6.C3-Bbaa1 mice, formally linking genetic elements within Bbaa1 to pathological production of type I IFN. Bone marrow-derived macrophages from Bbaa1 congenic mice implicated this locus as a regulator of type I IFN induction and downstream target gene expression. Bbaa1-mediated regulation of IFN-inducible genes was upstream of IFN receptor-dependent amplification; however, the overall magnitude of the response was dependent on autocrine/paracrine responses to IFN-β. In addition, the Bbaa1 locus modulated the functional phenotype ascribed to bone marrow-derived macrophages: the B6 allele promoted expression of M2 markers, whereas the C3H allele promoted induction of M1 responses. This report identifies a genetic locus physically and functionally linked to type I IFN that contributes to the pathogenesis of both Lyme and RA.

Figure 1. Interval specific congenic mice reveal contribution of C3H allele of Bbaa1 to Lyme arthritis severity

B6, C3H, B6.C3-Bbaa1, and C3.B6-Bbaa1 mice were infected with 2 × 10⁴ B. burgdorferi and arthritis was assessed at 4 wks. of infection, as described in Materials and Methods, and shown for (A) Change in ankle measurement and (B) Overall lesion score. Statistical significance of differences between ISCL and background parental strain were determined by Student’s t-test for ankle swelling and Mann Whitney U test for Overall lesion. All categories were negative for uninfected mice, injected with BSK media, and are not shown in the figure. Groups consisted of approximately equal numbers of male and female mice, with n=22 B6, 20 C3H, 23 B6.C3-Bbaa1, and 30 C3.B6-Bbaa1.

Figure 2. mAb blocking of Type I IFN signaling prevents the Bbaa1 dependent increase in arthritis in B. burgdorferi infected mice

B6.C3-Bbaa1 mice were treated with 2.5 mg MAR1-5A3 blocking mAb or isotype control the day before infection. Congenic and parental B6 and C3H mice were infected with B. burgdorferi and arthritis severity analyzed at 4 wks. of infection, shown for (A) Change in ankle measurement and (B) Overall lesion score. Statistical significance was determined by Student’s t-test for ankle swelling while the Mann Whitney U test was used for Overall lesion. n=5 per group.

Figure 3. Bone marrow derived macrophages (BMDM) reveal Bbaa1 regulates the magnitude of the IFN response to B. burgdorferi.

RT-PCR analysis of transcripts in BMDM from B6, C3H, B6.C3-Bba1 and C3.B6-Bbaa1 mice treated with media, sonicated B. burgdorferi (sonicate) or living B. burgdorferi (B. burgdorferi) for 6 hours. Transcript levels for Ifnβ, Gbp2, Cxcl9, Cxcl10, Iigp, and Tnfα were normalized to β-actin. Data are averages ± SE, with trends representative of 5 separate experiments. Significance determined by ANOVA, with Bonferroni post-hoc comparison, shown for ISCL with appropriate background parental strain. Differences between B6 and C3H for Ifnβ, Gbp2, Cxcl9, Cxcl10, and Iigp were also significant (not shown).

Figure 4. Phagocytic capacity of macrophages from B6 and C3H mice

Peritoneal macrophages (5 × 10⁵) were incubated with GFP-B. burgdorferi for 1 (A) and 2(B) hours at 37°C. Cells collected in the absence of trypsin (Total Cell Associated) or following incubation with trypsin (Trypsin Resistant) to remove extracellular bacteria. The percentage of macrophages with associated bacteria was determined by flow cytometry, as compared with cells not receiving bacteria. Data are averages ± SE, and are representative of 2 separate experiments. Significance determined by ANOVA, with Bonferroni post-hoc comparison; for B6 vs. C3H in each treatment group.

Figure 5. Bbaa1 regulates BMDM responses to poly I:C

RT-PCR analysis of transcript induction in BMDM from B6, C3H, B6-C3.Bba1 and C3.B6-Bbaa1 mice treated with poly I:C for 6 hours. Transcript levels for Ifnβ, Gbp2, and Iigp were normalized to β-actin. Data are averages ± SE, with trends representative of 2 separate experiments. Significance determined by ANOVA, with Bonferroni post-hoc comparison; shown for ISCL with appropriate background parental strain and for B6 vs. C3H.

Figure 6. Bbaa1 regulation of IFN profile is dependent on IFNAR feed forward

RT-PCR of transcript induction in BMDM from B6, C3H, B6-IFNAR1^−/−, and C3H-IFNAR1^−/− mice incubated with media, B. burgdorferi, or poly I:C for 6 hours. Transcript levels for Stat1, Stat2, Oasl2, Nos2. Tyki, Cxcl9, Cxcl10, IL-10, and Tnfα were normalized to β-actin. Significance determined by ANOVA, with Bonferroni post-hoc comparison; shown for B6 vs. B6-IFNAR1^−/− and C3H vs. C3H-IFNAR1^−/−.

Figure 7. Responses of BMDM to increasing doses of IFNβ

BMDM from B6 and C3H mice were treated with 0.4–100 Units/ml of IFNβ, and transcriptional responses measured at 3 hours. Transcript levels of Tyki, Nos2, Iigp, Arg1, Oasl2, and Mrc1 were normalized to β-actin. Data points indicate mean ± SE. Statistical significance between mouse strains were determined by Student’s t-test, as are indicated for Nos2 and Arg1.

Figure 8. Bbaa1 regulated levels of expression of M1 and M2 markers in resting and activated macrophages

BMDM from B6, C3H, B6.C3-Bbaa1 and C3.B6-Bbaa1 mice were assessed for M2 markers Arg1 and Mrc1 prior to addition of stimulant. Expression of the M1 marker Nos2 was assessed at 6 hours of stimulation with B. burgdorferi. All transcripts were normalized to β-actin. Shown are mean ± SE for one experiment, representative of 2 separate experiments. Significance determined by ANOVA, with Bonferroni post-hoc comparison; shown for ISCL with appropriate background parental strain and for B6 vs. C3H.

Figure 9. Impact of Bbaa1 on severity of K/B×N serum transfer arthritis

B6, B6.C3-Bbaa1, and C3H mice were treated with 100 μl K/B×N serum on Days 0 and 2, and arthritis was assessed by change in ankle measurement on Days 1–7 (A) and by histopathological scoring for overall lesion score on Day 7 (B), as described in Materials and Methods. Statistical significance was determined by Student’s t-test for ankle swelling and Mann Whitney U test Overall lesion. n=5

Figure 10. Enhanced rheumatoid arthritis in B6.C3-Bbaa1 mice is dependent on Type I IFN

B6.C3-Bbaa1 mice were treated with 2.5 mg MAR1-5A3 blocking mAb or isotype control the day before administration of K/B×N serum. B6.C3-Bbaa1 and B6 mice received 100 μl of K/B×N serum on Days 0 and 2. Arthritis was assessed by change in ankle measurement on Days 1–7 (A) and by histopathological scoring for Overall lesion score on Day 7 (B), as described in Materials and Methods. Statistical significance was determined by ANOVA, with Bonferroni post-hoc comparison for ankle measurements, and Kruskal-Wallis test with Dunn’s multiple comparison for Overall lesion. n=5.