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Infection and Immunity, April 2007, p. 2075-2078, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01920-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mice Lacking CD21 and CD35 Proteins Mount Effective Immune Responses against Borrelia burgdorferi Infection{triangledown}

Amanda C. Jacobson,1 Ying Ma,1 James F. Zachary,2 Janis J. Weis,1 and John H. Weis1*

Division of Cell Biology and Immunology, Department of Pathology, 15 North Medical Drive East, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650,1 Department of Veterinary Pathobiology, University of Illinois, Urbana, Illinois 618022

Received 5 December 2006/ Returned for modification 2 January 2007/ Accepted 25 January 2007


   ABSTRACT
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CD21/35–/– mice, deficient in CD21 and CD35 (complement receptors 2 and 1, respectively), were infected with Borrelia burgdorferi to assess the role of these receptors in a chronic bacterial infection. Although CD21/35–/– mice on both C57BL/6 and BALB/c backgrounds produced less B. burgdorferi-specific antibodies than did wild-type mice, spirochete levels and arthritis severity were similar.


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Complement plays an important part in the clearance of bacteria. The decoration of pathogen antigens with complement fragments provides signals important for the opsonization of bacteria and induction of inflammatory responses. In the mouse, CD21 and CD35 (complement receptors 2 and 1, respectively; CD21/35) are spliced products from the Cr2 gene and serve as receptors for complement breakdown products. On B cells, CD21/35 functions as the B-cell coreceptor and augments B-cell signaling (5). Mice lacking CD21/35 (CD21/35–/–) mount decreased antibody responses to T-cell-dependent and T-cell-independent antigens, with an impairment in the immunoglobulin G3 (IgG3)-specific antibody response (5). Consequently, CD21/35–/– mice were more susceptible to lethal Streptococcus pneumoniae infection (6). These studies reveal an important role for CD21/35 in protective antibody responses during acute bacterial infection; however, the role of these proteins during a chronic bacterial infection has not been addressed.

Infection of mice with the spirochete Borrelia burgdorferi provides an excellent model for understanding host-pathogen interactions involved in chronic infection. Arthritis caused by persistent infection of the spirochete within joint tissue appears to result from the sustained inflammatory challenges to the host from the bacterial components (18). Bacterium-specific antibodies produced during infection are not causative of or protective against acute Lyme arthritis in mice; however, they are important for controlling spirochete growth and resolution of disease (2, 14).

B. burgdorferi has evolved mechanisms to evade the host immune response, including resistance to host complement. B. burgdorferi is able to bind host complement regulatory proteins factor H and factor H-like 1, which permits the bacteria to evade complement-mediated killing (1, 4, 7-9, 13, 17). Additionally, higher spirochete levels and generally higher histopathology scores are found in joint tissue of C3–/– mice (10). In these mice, anti-B. burgdorferi IgG levels were reduced compared to those in the wild type. These data suggest a role for complement proteins and their receptors in the immune response against B. burgdorferi.

In this study, we used B. burgdorferi infection to assess the role of complement receptor proteins CD21 and CD35 in a chronic bacterial infection. CD21/35–/– mice were generated as previously described (6) and used at the 10th generation backcross to both C57BL/6 and BALB/c mice.

To assess the role of complement receptor proteins CD21 and CD35 in B. burgdorferi-specific antibody production, wild-type and CD21/35–/– animals on both the BALB/c and C57BL/6 backgrounds were infected intradermally with a cloned N40 isolate of B. burgdorferi that had been passaged three times (provided by S. Barthold, University of California, Davis). To achieve equivalent disease severity on both backgrounds, mice on the BALB/c background received 2 x 104 bacteria, whereas C57BL/6 mice received 2 x 103 bacteria (12). C3H mice were infected with 2 x 103 bacteria as positive controls for infection. Bacterium-specific and total antibody titers were measured (12) at 2 and 4 weeks postinfection. B. burgdorferi-specific IgG3 antibodies were significantly decreased at 4 weeks postinfection in CD21/35–/– animals on the C57BL/6 background, whereas a less drastic decrease was observed on the BALB/c background (Table 1). CD21/35–/– mice produced increased bacterium-specific IgM antibodies on the BALB/c background. Knockout (KO) animals on both strain backgrounds produced significantly less B. burgdorferi-specific IgG2b (Table 1). Collectively, CD21/35–/– mice had varied abilities to produce bacterium-specific antibodies important for immediate (IgM and IgG3) and long-term (IgG2b) protection. Total isotype-specific antibody titers in infected CD21/35–/– mice were similar to those of infected wild-type mice and increased greater than twofold relative to those of uninfected mice (data not shown). This demonstrates that the CD21/35–/– mice do not have an overall defect in the production of Ig against B. burgdorferi.


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  		TABLE 1. Effect of CD21/35 deficiency on B. burgdorferi-specific isotype distribution

 
The decoration of bacteria with complement fragments provides signals for the opsonization, lysis, and clearance of the bacteria. To determine if the antigens recognized by CD21/35–/– mice were altered relative to wild-type mice, Western blot analysis was performed comparing the antigens recognized by sera (3) from infected CD21/35–/– mice and wild-type controls. IgG and IgG3 in sera from CD21/35–/– and wild-type mice recognized similar proteins in B. burgdorferi lysates (Fig. 1). Sera from infected CD21/35–/– and wild-type mice were also analyzed for antibody against the C6 peptide of the VlsE variable lipoprotein of B. burgdorferi, which has been used diagnostically for Lyme borreliosis in humans (11). There were no significant differences in anti-C6 peptide antibody levels between wild-type and CD21/35-deficient mice on both the C57BL/6 and BALB/c backgrounds (data not shown). In brief, CD21/35–/– mice appear to recognize and mount antibody responses against a variety of B. burgdorferi antigens, including lipoproteins, albeit producing lower levels of B. burgdorferi-specific antibodies.


Figure 1
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  		FIG. 1. Diversity of antigens recognized by IgG and IgG3 in serum from infected CD21/35–/– mice. Western blot analysis of IgG (A) and IgG3 (B) antibodies directed against B. burgdorferi in sera diluted to 1:200 from infected BALB/c and CD21/35–/– mice sacrificed at 2 weeks or 4 weeks postinfection or in uninfected control sera. Similar results observed on a C57BL/6 background. OspA was detected with monoclonal OspA around 25 kDa, as indicated by prestained protein markers. OspC was detected with rabbit polyclonal OspC around 40 and 19 kDa. WT, wild type; Un-Inf, uninfected. The values on the left are molecular sizes in kilodaltons.

 
Bacterium-specific antibodies produced during infection play a role in controlling bacterial numbers and resolution of disease (14). To consider the effect of decreased B. burgdorferi-specific antibody responses in the CD21/35–/– mouse, spirochete numbers and arthritis severity of the rear ankle joints of infected animals were assessed by quantitative PCR as described in detail in references 15 and 19. Deficiency in CD21/35 did not have an effect on B. burgdorferi numbers in ankle tissue 4 weeks postinfection (Fig. 2), indicating that the antibodies produced by a CD21/35–/– animal are sufficient to control spirochete numbers. Spirochete numbers in the joint tissue of BALB/c mice correlates with arthritis severity (12). Arthritis severity within the ankle joints of infected mice did not differ significantly between wild-type and CD21/35-deficient mice as seen by ankle swelling and histological analysis (Table 2).


Figure 2
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  		FIG. 2. Spirochete levels in joint tissue of infected CD21/35–/– mice. B. burgdorferi DNA levels in the joints of infected C57BL/6 and CD21/35–/– mice 4 weeks postinfection were assessed by quantitative PCR. Similar spirochete levels were observed in BALB/c and CD21/35–/– mice, but they were lower than those in infected C57BL/6 or C3H animals.

 

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  		TABLE 2. Ankle pathology in CD21/35–/– mice infected with B. burgdorferi

 
Converse to the acute bacterial infection with S. pneumoniae, CD21/35–/– mice demonstrated the ability to control B. burgdorferi infection to wild-type levels regardless of an altered repertoire of antibodies against the bacteria. This may be best explained by the role of antibodies in protecting the host against bacterial infections. IgG3 is the major mouse IgG isotype produced in response to T-cell-independent type 2 antigens like pneumococcal polysaccharides (16). Conversely, the immune response against Borrelia antigens uses both innate antibodies, like IgG3 and IgM, and those isotypes which require T-cell help, like IgG2a/b (14). Complement proteins may be more critical adjuvants for immune responses against S. pneumoniae polysaccharides than B. burgdorferi lipoproteins. The evasion of host complement activation by the more recently described B. burgdorferi CRASP proteins may be more critical for bacterial survival during the initial infection. Complement receptors CD21/35, on the other hand, would have a more significant role in complement-mediated immune responses to the bacteria within the splenic environment, like antibody production. The data presented here suggest that complement receptors CD21 and CD35 do not have a direct role in regulating spirochete numbers, and the ability of CD21/35–/– mice to control B. burgdorferi infection suggests that B. burgdorferi does not have an advantage in a host lacking complement receptors 1 and 2.


   ACKNOWLEDGMENTS
 
This work was supported by U.S. Public Service grants AI-24158 to J.H.W. and AI-32223 to J.J.W. from the National Institutes of Health.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathology, University of Utah School of Medicine, 15 North Medical Drive East, Salt Lake City, UT 84112-5650. Phone: (801) 581-7054. Fax: (801) 585-7376. E-mail: john.weis{at}path.utah.edu. Back

{triangledown} Published ahead of print on 5 February 2007. Back

Editor: W. A. Petri, Jr.


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Infection and Immunity, April 2007, p. 2075-2078, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01920-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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