Borrelia burgdorferi-induced inflammation facilitates spirochete adaptation and variable major protein-like sequence locus recombination

Abstract

Spirochete adaptation in vivo is associated with preferential Borrelia burgdorferi gene expression. In this paper, we show that the administration of B. burgdorferi-immune sera to IFN-gammaR-deficient mice that have been infected with B. burgdorferi N40 for 4 days causes spirochete clearance. In contrast, immune sera-mediated clearance of B. burgdorferi N40 is not apparent in immunocompetent mice, suggesting a role for IFN-gamma-mediated responses in B. burgdorferi N40 host adaptation. B. burgdorferi-immune sera also induces clearance of B. burgdorferi N40 that have been passaged in vitro 75 times (B. burgdorferi N40-75), a derivative of B. burgdorferi N40 that does not rapidly adapt in vivo in immunocompetent mice. B. burgdorferi N40-75 produce lower levels of IFN-gamma and IL-12 in mice than does B. burgdorferi N40, and the administration of these cytokines to B. burgdorferi N40-75-infected mice results in an increased spirochetal burden, further indicating that IFN-gamma-mediated events promote B. burgdorferi survival. Differential immunoscreening and RT-PCR demonstrate that IFN-gamma-mediated signals facilitate spirochete recombination at the variable major protein like sequence locus, a site for early antigenic variation in vivo, and that recombination rates by B. burgdorferi N40 are lower in IFN-gammaR-deficient mice than in control animals. These results suggest that the murine immune response can promote the in vivo adaptation of B. burgdorferi.

FIGURE 1

Infection with B. burgdorferi N40-75 results in lower proinflammatory cytokine production and innate immune cell activation. A, Whole splenocytes from 3-wk-infected C3H mice were used to obtain total RNA. Expression of IFN-γ and the small subunit of IL-12 were analyzed by RT-PCR and hybridization with specific internal probes. HPRT (control) was included to assure equal loads of RNA. B, Expression of IL-4 was also analyzed by the same method. C, IFN-γ expression was measured in the joints of C3H mice infected with B. burgdorferi N40-75 or B. burgdorferi N40. Total RNA was extracted as described previously and analyzed by RT-PCR. HPRT (control) assured equal loads of RNA. D, The levels of IFN-γ and IL-12 were measured by ELISA in the sera of mice infected with B. burgdorferi N40-75 or B. burgdorferi N40 for 3 wk. Results represent a pool of sera from five mice and are representative of five individual experiments. E, CD4⁺ T cell restimulation in response to B. burgdorferi Ags. CD4⁺ T cells were purified from the spleens of 3-wk-infected C3H mice and restimulated in vitro with 10 µg/ml of a B. burgdorferi extract. The data presented are representative of three separate experiments with similar results. F, B. burgdorferi N40-75 and B. burgdorferi N40 extracts were used to activate innate immune cells in vitro. Whole splenocytes from C3H mice were stimulated for 48 h with 20 µg/ml spirochetal extracts and analyzed for IFN-γ production by capture ELISA.

FIGURE 2

IL-12 or IFN-γ increases Th1 responses and spirochetal burdens in B. burgdorferi N40-75-infected mice. Mice were treated with IL-12 (A, B, and C) or IFN-γ (D and E) and infected with B. burgdorferi N40-75. Control mice were infected with B. burgdorferi N40 or treated with the recombinant cytokines without subsequent infection. At the time of sacrifice, the amount of IFN-γ in the pooled sera of the mice was determined by capture ELISA (A andD). CD4⁺ T cells were purified from B. burgdorferi N40-75-infected, IL-12-treated mice, and controls and restimulated in vitro with B. burgdorferi extracts (B). The supernatants were analyzed for IFN-γ by capture ELISA. The relative amount of spirochetal DNA in the joints of the infected mice was determined by PCR with primers corresponding to the flaB gene (C and E). Equal amounts of DNA were used to perform the PCR (500 ng/reaction). All results are representative of at least two independent experiments.

FIGURE 3

Comparison of the sequences of the four clones identified by differential immunoscreening. Sera from mice infected by syringe (nonadapted spirochetes) and transplant (host-adapted spirochetes) were used to probe a B. burgdorferi expression library. Clones that selectively reacted with sera from mice infected with the host-adapted spirochetes were isolated. Gray boxes indicate identical residues. Gaps in the sequence are indicated by dashes.

FIGURE 4

Recombination is impaired in IFN-γRα-deficient mice. The 129/SvJ and IFN-γRα-deficient mice were infected with clonal B. burgdorferi B31, and 4 days later, the skin at the site of inoculation was incubated in BSK II medium. DNA was then extracted and amplified using primers flanking the variable domain of the vlsE gene. Only nucleotide changes are noted. Gaps in the sequence are marked with spaces. The six variable regions within the variable domain are boxed. These data represent cumulative changes found in 19 (129/SvJ mice) and 11 sequences (IFN-γRα-deficient mice), respectively.