Infect. Immun. March 2008 76:3 1153-1162;
Although it is capable of eliciting strong innate and adaptive immune responses, Borrelia burgdorferi often evades immune clearance through largely unknown mechanisms. Our previous studies determined that infected interlukin-10−/−
) mice show significantly lower B. burgdorferi levels than wild-type (B6) mice and that IL-10 inhibits innate immune responses critical for controlling B. burgdorferi infection. To determine whether virulent B. burgdorferi preferentially enhances IL-10 production, we developed an in vitro coculture medium (RPMI.B) in which both B. burgdorferi and primary macrophages
(Mφs) remain viable. B. burgdorferi grew at similar rates and was able to regulate expression of immunoreactive proteins with similar kinetics in RPMI.B and in traditional BSK medium; in contrast, B. burgdorferi cultured in conventional tissue culture medium (RPMI) rapidly lost viability. Coculture of viable B. burgdorferi in RPMI.B with Mφs resulted in more rapid and significant increases in IL-10 transcripts and secreted proteins than coculture with nonviable B. burgdorferi in RPMI, which corresponded with decreased production of proinflammatory cytokines
. Addition of live B. burgdorferi to Mφs in RPMI.B also elicited substantially higher IL-10 levels than heat-killed bacteria elicited, confirming that increased IL-10 production was not inherent to coculture in RPMI.B. Transfer of supernatants from B. burgdorferi-stimulated Mφs into naïve Mφ cultures resulted in suppressed activation upon subsequent stimulation with different bacterial agonists, and this suppression was obviated by IL-10-specific antibody. in vivo
analyses determined that murine skin samples exhibited substantial upregulation of IL-10 within 24 h of injection of B. burgdorferi. Together, these results suggest that viable B. burgdorferi can suppress early Mφ responses during infection by causing increased release of IL-10.
Infection of susceptible hosts with strains of Borrelia burgdorferi sensu lato causes Lyme disease, which accounts for the majority of vector-borne illness in the United States and most of the temperate regions of Europe and Asia (18, 41). Upon introduction into the host dermis by an infected tick, the spirochetal bacteria must quickly adapt to their vertebrate host, disseminate from the skin to various host tissues, and evade the developing immune responses. This evasion must continue until the bacteria receive appropriate signals that lead to reemergence from their immunoprivileged niche and subsequent migration to and infection of a feeding tick. Although this extended persistence within an immunocompetent host is essential for the existence of the bacteria, the mechanisms that they utilize to escape immune clearance are largely unknown.
Infection with B. burgdorferi triggers strong innate and adaptive immune responses that are largely directed against the ≥127 putative lipoproteins believed to be produced by this spirochete (8, 12, 37). All of these proteins possess a signal II peptidase sequence which can be used to predict their subsequent cleavage and the triacyl modification of the resultant N-terminal cysteine residue. The triacylated lipoproteins are highly immunogenic, as injection of prototypic lipoproteins elicits strong antibody responses and sera collected from infected individuals possess high levels of antibodies that are specific to varioous lipoproteins (31, 45). Additionally, these triacylated lipoproteins directly activate macrophages
(Mφs) (30), dendritic cells (35, 42), neutrophils (28), endothelial cells (48, 49), mast cells (24), fibroblasts (11), and B lymphocytes
(23) through signaling events mediated by Toll-like receptor 2 (TLR2) (16, 47, 50). Since B. burgdorferi either lacks many of the known bacterial agonists
that have stimulatory activities (e.g., lipopolysaccharide
[LPS] and lipoteichoic acid) or sequesters them so that they cannot be readily accessed by host immune mediators (e.g., endoflagella), it appears that borrelial lipoproteins are a major focus of both innate and adaptive immune responses to this spirochete. The importance of these interactions is reflected by the relative inability of Mφs derived from TLR2−/−
mice to respond to B. burgdorferi and/or its lipoproteins, as well as the high levels of B. burgdorferi that persist in target tissues of infected TLR2−/−
mice compared to wild-type mice (47). Interestingly, TLR2−/−
mice produce a B. burgdorferi-specific antibody response that appears to be almost identical to that produced by wild-type mice (47, 52), and passive transfer of B. burgdorferi-elicited immune serum from TLR2−/−
mice and passive transfer of B. burgdorferi-elicited immune serum from wild-type mice protect naïve mice from infection equally (52), suggesting that appropriate innate immune responses are the most critical responses for bacterial clearance.
The importance of the innate immune responses, particularly Mφs, for controlling B. burgdorferi infection is also reflected by the results of multiple studies assessing the immunosuppressive effects of interleukin-10
(IL-10) on the development of Lyme disease. IL-10 is a significant immunomodulatory cytokine, largely due to its broad but potent anti-inflammatory properties (27). Although IL-10 can be produced by and act upon many cell types, myeloid cells are reported to be the major source of IL-10 production (15). Many of the suppressive immune functions generally associated with IL-10 activities are also mediated through myeloid cells, either by direct downregulation of their production of proinflammatory mediators or by inhibition of their ability to mediate critical antigen
-presenting functions to lymphocytes. Exposure of human and murine Mφs to B. burgdorferi elicits significant IL-10 production (7, 13, 14, 29), and addition of exogenous IL-10 to Mφs can suppress the production of proinflammatory mediators produced in response to B. burgdorferi (7). Infection studies have indicated that IL-10−/−
mice are significantly better at clearing B. burgdorferi from target tissues than wild-type mice. Infected IL-10−/−
mice produce higher levels of B. burgdorferi-specific antibodies than wild-type mice, but the differences are not directly responsible for the enhanced bacterial clearance that occurs (19). IL-10−/−
mice also exhibit enhanced innate immune barriers to B. burgdorferi infection compared to wild-type mice, and IL-10−/−
Mφs exposed to spirochetes in vitro produce higher levels of multiple proinflammatory mediators than wild-type Mφs (19). Based on these studies, evasion of early innate responses appears to be critical for efficient dissemination and persistence of B. burgdorferi, and suppression of these innate responses by IL-10 is detrimental to effective immune clearance.
Recently, several bacterial and viral pathogens have been shown to promote elevated IL-10 production, to suppress various host immune functions, and to subsequently evade immune clearance (26, 33), raising the possibility that virulent B. burgdorferi might also possess mechanisms that preferentially induce Mφs to upregulate IL-10 production. Because most in vitro analyses of mammalian immune cells are performed in tissue culture medium that does not permit the growth or survival of this fastidious spirochete, it is unlikely that B. burgdorferi could exhibit such a putative virulence mechanism using traditional mammal-based culture conditions. We therefore first developed an in vitro coculture medium that maintains the viability of this fastidious bacterium but does not inherently activate murine Mφs. Subsequent studies using this optimized coculture system indicated that viable B. burgdorferi does potently elicit rapid IL-10 induction by Mφs, and the IL-10 then acts in an autocrine loop to suppress appropriate inflammatory responses necessary to promote efficient immune clearance.