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The Journal of Immunology, 2003, 170: 3819-3827.
Copyright © 2003 by The American Association of Immunologists

The Resolution of Relapsing Fever Borreliosis Requires IgM and Is Concurrent with Expansion of B1b Lymphocytes1

Kishore R. Alugupalli2,*, Rachel M. Gerstein*, Jianzhu Chen{ddagger}, Eva Szomolanyi-Tsuda{dagger}, Robert T. Woodland* and John M. Leong*

Departments of * Molecular Genetics and Microbiology and {dagger} Pathology, University of Massachusetts Medical School, Worcester, MA 01655; and {ddagger} Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The rate of pathogen clearance is a critical determinant of morbidity and mortality. We sought to characterize the immune response responsible for the remarkably rapid clearance of individual episodes of bacteremia caused by the relapsing fever bacterium, Borrelia hermsii. SCID or Rag-/- mice were incapable of resolving B. hermsii infection, indicating a critical role for T and/or B cells. TCR-/- mice, which lack T cells, and IL-7-/- mice, which are deficient in both T cells and follicular B cells, but not in B1 cells and splenic marginal zone (MZ) B cells, efficiently cleared B. hermsii. These findings suggested that B1 cells and/or MZ B cells, two B cell subsets that are known to participate in rapid, T-independent responses, might be involved. The efficient resolution of the episodes of moderate level bacteremia by splenectomized mice suggested that MZ B cells do not play the primary role in clearance of this bacterium. In contrast, xid mice, which are deficient in B1 cells, suffered more severe episodes of bacteremia than wild-type mice. The hypothesis that B1 cells are critical for clearance of B. hermsii was further supported by a selective expansion of the B1b (i.e., IgMhigh, IgD-/low, Mac1+ CD23-, and CD5-) cell subset in infected xid mice, which coincided with the eventual resolution of infection. Finally, mice selectively incapable of secreting IgM, the dominant isotype produced by B1 cells, were completely unable to clear B. hermsii. Together these results support the model that B1b cells generate the T-independent IgM required for the control and resolution of relapsing fever borreliosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Relapsing fever, caused by several species of Borrelia, such as B. hermsii, B. turicatae, and B. duttonii, provides a natural infection model with which to study the clearance of bacteria from the bloodstream. Although relapsing fever spirochetes can infect a number of organs, they remain predominantly localized in the blood, causing multiple episodes of high level (103–105 spirochetes/µl) bacteremia (1, 2). Each of these episodes is associated with the outgrowth of spirochetes of a different serotype, the specificity of which is determined by the major surface Ag, the variable major protein (Vmp).3 DNA rearrangements involving silent vmp genes and a single expression locus allow the spirochete to express any one of many Vmps, and each episode of bacteremia is caused by spirochetes expressing an antigenically distinct Vmp (3). Interestingly, each episode of B. hermsii bacteremia is cleared quite rapidly, i.e., within 2–3 days (4, 5). Given that a variety of microbial pathogens use similar mechanisms of antigenic variation to evade the host immune system, and that such mechanisms pose a challenge for the development of effective vaccines (6), it is of interest to understand the mechanism(s) by which this remarkably rapid clearance occurs.

It has been known for many years that Abs are critical for eliminating relapsing fever borreliae. Comparison of infection by two species of relapsing fever spirochete reveals a temporal correlation between bacterial clearance and the induction of an Ab response to spirochete Ags (4). The onset of B. duttoni bacteremia is delayed by passive transfer of serum from infected mice, and the induction of this protective response coincides with bacterial clearance during acute infection (7). The observation that nude (8) or neonatally thymectomized (9) mice clear relapsing fever spirochetes indistinguishably from wild-type (wt) mice suggests that this Ab response is independent of T cells. Nevertheless, because nude mice retain small numbers of functional CD4+ and CD8+ cells (10), and nude and thymectomized mice have {gamma}{delta} T cells of extrathymic origin (11), these studies do not rule out a role for T cells in the resolution of relapsing fever borreliosis.

IgM, one of the isotypes associated with T-independent responses, appears to be an important effector molecule for eliminating relapsing fever borreliae (7, 8, 12, 13). Polyclonal IgM isolated from mice infected with relapsing fever spirochetes confers passive protection (7, 12, 13), as does a monoclonal IgM derived from a B. hermsii-infected mouse (8). Although IgG subclasses, particularly IgG3, also confer passive protection, they are generated later in the infection (12), and the production of IgM, but not IgG, temporally correlates with clearance of the Spanish relapsing fever spirochete (13). Consistent with an important role for IgM in clearance of relapsing fever spirochetes, infection of SCID (14, 15) or B cell-deficient (13) mice with relapsing fever spirochetes results in persistent bacteremia. Nevertheless, genetic evidence that IgM in particular plays an essential role during clearance of an active infection is currently lacking.

Although B cells are clearly required for elimination of relapsing fever bacteremia, the roles of specific B cell subsets in this clearance process have not yet been defined. B cells residing in the mature, long-lived pool are heterogeneous (16). Follicular (FO) B cells, which comprise the majority of total B cells in the body, recirculate among the B cell-rich lymphoid follicles, whereas marginal zone (MZ) B cells are extrafollicular, relatively sessile, and localized to the marginal sinus of the spleen (16). FO and MZ B cells collectively comprise the B2 subclass, commonly referred to as conventional B cells. In contrast, B1 cells are most abundant in peritoneal and pleural cavities and can be subdivided into B1a (CD5+) and B1b (CD5-) subsets (17). Although B1 and MZ B cells are of distinct B cell subsets, they share several functional characteristics (16, 18). For example, both B1 and MZ B cell types maintain normal numbers throughout life after arrest of B lymphopoiesis, suggesting their self-renewal capability (19, 20). Furthermore, both B1 and MZ B cells are good responders to T-independent Ags (16, 18, 21). Indeed, it has recently been shown that MZ and B1 cells mount a coordinated response to blood-borne, T-independent particulate Ags (22). Finally, both MZ B and B1 cells mount rapid IgM responses (16, 21), a particularly interesting property given the rapid clearance of spirochetes from the blood during relapsing fever and the suggestion that IgM plays a critical role in this process (7, 8, 12, 13).

To better understand the mechanism of rapid clearance of relapsing fever spirochetes from the blood, we have examined B. hermsii infection in mice deficient in T, B2, or B1 cells; in splenectomized mice lacking MZ B cells; and in mice incapable of producing secretory IgM. We found that a T-independent secretory IgM response is essential for the elimination of active B. hermsii infection. We also found a correlation between the specific expansion of B1b cells and the resolution of bacteremia. These results are consistent with the hypothesis that this B cell subset generates the IgM required for eliminating B. hermsii.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Borrelia strains

B. hermsii strain DAH, originally isolated from a relapsing fever patient northwest of Cheney, WA (23), was harvested from the blood of an experimentally infected mouse and passaged only once in BSK-H medium (Sigma-Aldrich, St. Louis, MO) containing 6% heat-inactivated rabbit serum (Sigma-Aldrich) at 33°C (referred to as DAH-p1). The strain DAH-p14 or DAH-p19 was generated by serial passage of the above strain (DAH-p1) in the same medium for 13 or 18 times, respectively, as previously described (5). We have previously reported that this repeated passage in vitro results in a less virulent phenotype of B. hermsii as determined by significantly diminished bacteremia (5).

Mice and infections

Mice housed in microisolator cages with free access to food and water were maintained in a specific pathogen-free facility of the Department of Animal Medicine at University of Massachusetts Medical School (UMMS). Normal or splenectomized 4-wk-old C57BL/6J, C57BL/6J-Rag1tm1 Mom, and C57BL/6J-PrkdcSCID mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice that lack the secreted form of IgM but have the membrane-bound form of IgM (sIgM-/-) have been previously described (24). These sIgM-/- mice are on a 129Sv background; a stock of control wt (129Sv) mice were purchased from The Jackson Laboratory and were bred at UMMS. TCR {beta}-chain-deficient (TCR-{beta}-/-) and TCR-{beta}-chain and TCR-{delta}-chain-deficient (TCR-{beta} x -{delta}-/-) mice on a C57BL/6 background (25, 26) were from the colony of Dr. E. Szomolanyi-Tsuda (UMMS) and were originally obtained from Dr. S. Tonegawa (Massachusetts Institute of Technology, Cambridge, MA). The 129Sv/BL6 mice were provided by Dr. R. Hynes (Massachusetts Institute of Technology). The IL-7-deficient mice (IL-7-/-) (27) on a mixed background of 129Sv/BL6 were from the colony of Dr. R. M. Gerstein (UMMS) and were originally obtained from Dr. R. Murray (DNAX Research Institute, Palo Alto, CA). CBA/Ca and CBA/N (xid; X-linked immunodeficient) mice were purchased from National Cancer Institute (Fredrick, MD).

Mice were infected i.v. at 6–12 wk of age unless stated otherwise. Infection with B. hermsii strain DAH-p1 in the dose range of 102–106 spirochetes results in bacteremia of similar degree and duration; however, the onset of bacteremia is directly proportional to the infecting dose (K. R. Alugupalli, unpublished observations). In the present study mice were infected with (2 x 105–5 x 105) spirochetes, and the bacteremia was monitored daily by darkfield microscopy (5).

Preparation of DNA from infected organs

Mice infected with DAH-p1 were sacrificed by CO2 asphyxiation and were transcardially perfused with ~50 ml of sterile saline (1–2 ml/min), to minimize the blood-derived B. hermsii contamination of the organs. The bladder, brain, heart, kidneys, liver, lungs, and spleen were excised, and total DNA from the organs was extracted as previously described with a slight modification (28). In brief, the tissues were minced and placed in individual 15-ml polypropylene tubes containing 2.5 ml of a 0.1% collagenase A (Roche, Indianapolis, IN) solution in sodium PBS (pH 7.4). Collagenase digestion was performed for 4 h at 37°C and then mixed with an equal volume of 0.2 mg/ml proteinase K (Roche) in 200 mM NaCl, 20 mM Tris-HCl (pH 8.0), 50 mM EDTA, and 1% SDS, and the digestion was continued for an additional 16 h at 55°C. DNA was extracted by adding an equal volume of phenol-chloroform. To maximize the DNA recovery from the extraction mixture we used Phase Lock Gel tubes (Eppendorf Scientific, Westbury, NY) according to the manufacturer’s instructions. DNA thus recovered was precipitated with ethanol and digested with 1 mg/ml DNase-free RNase (Sigma-Aldrich), the DNA samples were extracted twice with phenol-chloroform using the Phase Lock Gel tubes, ethanol precipitated, and resuspended in TE buffer (0.5 mM EDTA and 5 mM Tris-HCl (pH 7.5)). The DNA content was measured by OD at 260/280 nm (ratio, >=1.8) and adjusted to 40 µg/ml.

Measuring the amounts of B. hermsii flaB and mouse nidogen sequences by quantitative PCR

PCR was performed in a fluorescence temperature cycler (LightCycler, Roche). Amplification was performed in a 10-µl final volume containing 3 mM MgCl2, 50 mM Tris (pH 8.3), 0.05% BSA, 200 µM of each deoxynucleoside triphosphate, 0.015% SYBR Green I (Molecular Probes, Eugene, OR), 1 µM of each primer, 0.5 U of Taq polymerase, 110 ng of TaqStart Ab (Clontech, Palo Alto, CA), and 40 ng of template DNA. The oligonucleotide primers used to detect B. hermsii flaB were KA119.F (5'-CTG ATG ATG CTG CTG GTA TGG GCG TTG CTG-3') and KA460.R (5'-TGA TGC TGG TGT GTT AAT TTT TGC GGG TTG-3'). The oligonucleotide primers used to detect mouse nidogen were nido.F (5'-CCA GCC ACA GAA TAC CAT CC-3') and nido.R (5'-GGA CAT ACT CTG CTG CCA TC-3') (29). The standard amplification program included 40 cycles. Each cycle was comprised of heating at 20°C/s to 95°C with a 1-s hold, primer annealing at 70°C (for nidogen) and 72°C (for flaB) for 10 s, primer extension at 72°C for 30 s, and heating at 1°C/s to 80°C to acquire the specific fluorescence of the PCR product in each cycle. After amplification, a melting curve was acquired by heating the PCR product at 20°C/s to 95°C, cooling it at 20°C/s to 60°C, and slowly heating it at 0.2°C/s to 94°C with fluorescence collection at 0.2°C intervals. Melting curves were used to determine the specificity of the PCR (30).

Flow cytometry

Peritoneal cells (PerC) harvested from individual mice were counted, and the cell density was adjusted to 2.5 x 107/ml in staining medium (deficient RPMI medium 1640 (Irvine Scientific, Santa Ana, CA) with 3% new calf serum, 1 mM EDTA). After blocking the FcRs with 2.4G2 Ab (1 µg/106 cells), an aliquot of 25 µl of PerC was incubated in a microtiter plate with appropriately diluted Ab. Abs IgM-FITC (clone 1B4B1), IgD-PE or biotin (clone 11-26), Mac-1PE or biotin (clone M1/70), and CD5 PE or biotin (clone 53-7.3) were purchased from eBioscience (San Diego, CA), and streptavidin-PE-Cy5 was obtained from BD PharMingen (San Diego, CA). After staining, cells were washed twice with staining medium, and propidium iodide (1 µg/ml) was added to identify and exclude dead cells from subsequent analysis. These preparations were run on a FACSCalibur (BD Biosciences, Mountain View, CA) using CellQuest software for acquisition of the data (BD Biosciences). Data was analyzed using FlowJo software program (Treestar, San Carlos, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adaptive immunity is required for clearance of B. hermsii

Infection of wt mice with B. hermsii strain DAH passage 1 (DAH-p1) resulted in recurrent episodes of bacteremia (Fig. 1), consistent with previous experimental murine infections with several species of relapsing fever bacteria (4, 5, 7, 8, 9, 12, 13). These wt mice eventually cleared the bacteria from the blood (Fig. 1). Furthermore, bacterial DNA could not be detected by PCR in the heart, brain, spleen, or testis of animals 4 wk after infection (data not shown). In contrast, B. hermsii infection in SCID or Rag-/- mice resulted in a high level bacteremia that persisted continuously for 4 wk (Fig. 1). At this time the infected SCID mice were moribund, and the animals were sacrificed. Our data are consistent with previous studies of infections of SCID mice with B. turicatae (14) and B cell-deficient mice with the Spanish relapsing fever spirochete (13), and confirm that adaptive immunity is required for the clearance of B. hermsii as well.



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  		FIGURE 1. An adaptive immune system is required for the clearance of B. hermsii. C57BL/6J (wt; n = 5), C57BL/6J-Rag1tm1 Mom (Rag-/-; n = 5), or C57BL/6J-PrkdcSCID (SCID; n = 4) mice were infected i.v. with 4 x 105 B. hermsii DAH-p1. Bacteremia was measured by microscopic counting. For clarity, one representative bacteremia of a wt mouse was shown. The broken line indicates the detection limit for bacteremia.

 
Clearance of B. hermsii is T cell independent

The requirement for adaptive immunity for clearance of B. hermsii suggested that T cells, B cells, or both play an essential role in this process. To assess the role of T cells in protection, TCR-{beta}-/- mice (that lack {alpha}{beta} T cells) or TCR-{beta}-/- x -{delta}-/- mice (that lack both {alpha}{beta} and {gamma}{delta} T cells) were infected with B. hermsii DAH-p1. As assessed by the peak bacterial density and the duration of the first bacteremic episode (Table I) as well as subsequent episodes (data not shown), infection and clearance of these T cell-deficient mice were indistinguishable from infection of wt mice. These data indicate that the clearance of B. hermsii was T cell independent, consistent with previous reports on infections of nude or thymectomized mice (8, 9).


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  		Table I. T cells are not required for clearing B. hermsii bacteremia

 
FO B cells are not required for the clearance of B. hermsii

Given that it has recently been shown that mice deficient in B cells are unable to clear relapsing fever spirochetes (13), the above data suggested that T-independent B cell responses are sufficient to eliminate B. hermsii. Among the B cell subsets, B1 and MZ B are known to participate in T-independent responses, while FO B cells are critical for T-dependent responses.

IL-7 is a cytokine essential for lymphocyte development, and IL-7-/- mice are severely lymphopenic and deficient in FO B and T cells (27). The recent characterization of IL-7-/- mice by Carvalho and colleagues (19) showed a virtual arrest of B lymphopoiesis by the age of 8 wk, yet the MZ B cell and the B1 cell compartments are intact throughout the life of IL-7-/- mice. IL-7-/- mice therefore allow us to assess the relative importance of FO B cell vs MZ B and B1 cell responses in B. hermsii clearance. We found that the kinetics of clearance of each bacteremic episode of DAH-p1 in 12-wk-old IL-7-/- mice were comparable to those in wt mice (Fig. 2), indicating that FO B cells are dispensable for eliminating B. hermsii.



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  		FIGURE 2. FO B cells are not required for the clearance of B. hermsii. 129Sv/BL (IL-7+/+; n = 5) or IL-7-/- (n = 5) mice were infected i.v. with 2 x 104 B. hermsii DAH-p1. Bacteremia was measured by microscopic counting. The duration peak density and duration of the first as well as the second episodes of bacteremia were not statistically significant (by Student’s t test). Each plot represents bacteremia in an individual mouse. The broken line indicates the detection limit for bacteremia. The data are representative of two separate experiments.

 
A role for the spleen in the bloodstream clearance of B. hermsii is revealed only during high level, but not moderate level, bacteremia

The observation that FO B cells are not required for the elimination of B. hermsii suggests that MZ B and/or B1 cells play a critical role in this process. To determine whether MZ B cells, which reside exclusively in the MZ of the spleen, are required for clearance of B. hermsii, we performed infection studies with splenectomized mice. We previously showed that the ability of B. hermsii to cause high level (e.g., 104–105/µl) bacteremia was attenuated upon in vitro culture, such that a moderate passage B. hermsii strain DAH (e.g., passages 14–19), although still infectious, was only capable of growth to ~103/µl in the blood of infected mice (5). Although the putative genetic lesion(s) responsible for the diminished virulence of moderate passage B. hermsii is not defined, low and moderate passage strains provide a test system to explore the immune clearance mechanisms that may operate under different bacteremic loads. Thus, to examine the role of the spleen in conditions of either moderate or higher level bacteremia, we infected splenectomized or control mice with strain DAH-p19 or DAH-p1, respectively. Infection with the moderate passage strain (i.e., DAH-p19) caused bacteremic episodes of indistinguishable duration and severity in both splenectomized and unsplenectomized mice (Table II), indicating that MZ B cells are not required for the clearance of this strain and implying that B1 cells may play the more critical role. In contrast, the spleen apparently plays a significant role in controlling infection by B. hermsii DAH-p1, the strain that causes high level bacteremia: the first episode of bacteremia was more severe and of longer duration in splenectomized compared with control mice (Table II). The second episode of bacteremia after infection with B. hermsii DAH-p1 is typically less severe than the first (5), and no differences in density or duration of the second episode were observed between splenectomized and control mice, consistent with the moderate bacteremia results of strain DAH-p19 (Table II).


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  		Table II. B. hermsii bacteremia in splenectomized mice

 
Impaired clearance of B. hermsii in xid mice

The observation that the spleen, the organ in which MZ cells reside, is not required for the clearance of B. hermsii DAH-p19 suggested that B1 cells may play the primary role in elimination of this bacterium. Bruton’s tyrosine kinase (Btk) is important in B cell development and function (31, 32), and xid (CBA/N) mice, which have a point mutation in the Btk gene, are severely (>97%) deficient in B1 cells (31, 32). In contrast, their MZ cell population (33) is normal, and their number of FO B cells is ~50% that of wt mice (31, 32). As a result, xid mice respond normally to many T-dependent Ags, but poorly to T-independent type 2 Ags, such as polysaccharide Ags and Ags with repetitive structures, which are more dependent on Btk for B cell activation (31, 32, 34).

To evaluate the role of B1 cells in the control of B. hermsii infection, we infected wt and xid mice with the moderate passage strain, DAH-p14. The peak spirochete densities during episodes of bacteremia were 15- to 23-fold higher in xid mice than in wt mice, and the episodes persisted longer (p < 0.05; Fig. 3, A and B). This defect in bacterial clearance by xid mice was also apparent after infection with the low passage strain, B. hermsii DAH-p1 (Fig. 3, C and D). Although there was considerable mouse-to-mouse variation, the peak concentration of strain DAH-p1 in the first episode was at least 4-fold higher in xid mice than in wt mice, and again the episodes persisted longer (p < 0.05; Fig. 3, C and D). While the second peak of bacteremia caused by strain DAH-p1 was not affected by splenectomy (see Table II above), this peak of bacteremia in xid mice was dramatically more severe than that in wt mice (Fig. 3, C and D). Thus, infection by both the low and moderate passage DAH strains indicates that xid mice are significantly impaired in eliminating blood-borne B. hermsii, suggesting that B1 cells play a critical role in clearance.



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  		FIGURE 3. Impaired clearance of B. hermsii in xid mice. CBA/Ca (wt; n = 3) or CBA/N (xid; n = 5) mice, which are severely deficient in B1 cells, were infected i.v. with 4 x 105 B. hermsii DAH-p14 (A and B) or DAHp-1 (C and D). Bacteremia was measured by microscopic counting. The durations of both the first and second episodes of bacteremia in xid mice were significantly prolonged compared with those in wt mice (A and C; *, p < 0.05, by Student’s t test). Each plot represents bacteremia in individual mouse. The mean ± SD values for each group are also shown in the upper right corner. The broken line indicates the detection limit for bacteremia. The data are representative of two separate experiments. The peak density of bacteremia in xid mice was also significantly severe (B and D; p < 0.05, by Mann-Whitney test). Due to the high mouse-to-mouse variation, these data were analyzed by Mann-Whitney test (one-tailed).

 
Selective expansion of B1b cells in B. hermsii-infected mice

Although xid mice were clearly impaired in bacterial clearance (Fig. 3), by 4 wk postinfection bacteremia was apparently completely resolved (data not shown). In contrast, infection of SCID mice resulted in unremitting bacteremia (Fig. 1). These results indicate that xid mice are not totally incapable at eliminating bacteria, an apparent paradox if B1 cells are indeed critical for controlling bacteremia. One possible explanation is that the markedly reduced B1 population of xid mice expands during the infection. In fact, Kearney and colleagues (22) have shown that Ag-specific clones of B1 cells expand in wt mice immunized with heat-killed Streptococcus pneumoniae.

Given that B1 cells are found in abundance in the peritoneal cavity in wt mice, we analyzed the frequencies of peritoneal B cells to detect a potential B1 cell expansion in xid mice. Remarkably, the frequency of B1 cells (defined by the surface markers IgMhigh, IgDlow, Mac1+) in xid mice at 4 wk postinfection was 35-fold greater than that in uninfected xid mice (Fig. 4A, upper panel) and comparable to the frequency of B1 cells in uninfected wt mice (Fig. 4B, upper panel). This expansion occurred regardless of whether the mice were infected with a low or moderate passage strain (Fig. 4A, middle and right panels), consistent with a role for B1 cells in the control of infection by both of these strains. The increased frequency of the B1 subset in infected xid mice was not due to a diminution of other B cell subsets, but, rather, to an increase in the absolute numbers of B1 cells from a few hundred cells to ~105 cells in the peritoneal cavity (Fig. 5B). The expanded B1 population lacked CD5, the classical B1a cell marker, indicating that these cells phenotypically belong to the B1b subset (Fig. 4). The virtual lack of B1a cells in xid mice either pre- or postinfection (Figs. 4 and 5) suggests that this subset is not required for elimination of B. hermsii and indicates that B. hermsii infection elicits a strikingly specific expansion of the B1b subset. No expansion of B1 cells was noted in the spleens of xid or wt mice (data not shown).



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  		FIGURE 4. Increased frequency of the B1b cell population in B. hermsii-infected mice. Peritoneal cells of uninfected or 4-wk postinfected xid (A; CBA/N) or wt (B; CBA/Ca) mice were harvested and stained with Abs specific for IgM, IgD, and Mac1 or CD5 and analyzed by flow cytometry. All B cells were first identified by IgD and IgM dual positivity (plots not shown) and were further resolved as B1 (i.e., B1a and B1b) and B1a populations resolved by Mac1 and CD5 positivity, respectively. The percent frequency values of B1 and B1a cells among all PerC cells were indicated within the plots. The frequency of B1b cells was inferred from values obtained from the subtraction of the percent B1a (CD5+) from the percentage of all B1 cells (Mac1+). The data were generated by analyzing a minimum of 20,000 cells and are representative of three separate experiments. Five percent contour plots are shown.

 


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  		FIGURE 5. Selective expansion of B1b cell population in B. hermsii-infected mice. Peritoneal cells of uninfected (n = 3) or 4-wk postinfected CBA/N (xid; n = 3) or CBA/Ca (wt; n = 3) mice were harvested and stained with Abs specific for IgM, IgD, and Mac1 (CD11b) or CD5. The percent frequencies of cells (A) or the absolute cell counts (B) of B2 (IgDhigh, Mac1-) B1a (IgMhigh, IgDlow, Mac1+, CD5+), and B1b (IgMhigh, IgDlow, Mac1+, CD5-) were identified by flow cytometry. The mean ± SD values of respective subsets are given. Significant expansion of B1b cells occurred in xid mice infected with DAH-p1 (**, p < 0.002) and DAH-p14 (*, p < 0.02). The percent frequency of the B1b lymphocyte subset of wt mice infected with DAH-p1, but not DAH-p14, was also statistically significant (p < 0.002). The data are representative of two separate experiments.

 
Wt mice, which possess much higher baseline levels of B1b cells than xid mice and clear B. hermsii much better, also underwent a modest (2-fold) expansion of B1b cells after infection with DAH-p1 (Fig. 5, lower panels). No expansion was detectable in wt mice infected with strain DAH-p14 (Fig. 4B, right panel). Thus, the number of B1b cells at 4 wk postinfection generally correlated with bacterial burden; xid mice infected with DAH-p1 or p-14, or wt mice infected with DAH-p1, all suffered high level bacteremia and experienced significant expansion of B1b cells (Fig. 5). Wt mice infected with DAH-p14 suffered only moderate level bacteremia and demonstrated no significant increase in B1b cells. These results are consistent with the idea that specific B1 cells clones proliferate in response to a threshold level of an appropriate T-independent Ag (22).

Secretory IgM is essential for eliminating B. hermsii

Our data suggest that B1 cells are required for efficient clearance of B. hermsii, and that MZ B cells may also contribute to clearance when the bacterial load is high. IgM is the most dominant isotype in T-independent responses and is produced by B1 and MZ B cells (16). In addition, transfer of the IgM fraction of immune serum was shown to prevent the onset of bacteremia by the relapsing fever spirochetes when administered before the bacterial challenge (7, 12, 13). To unambiguously test whether IgM is required for B. hermsii clearance during active infection, we infected sIgM-/- mice, which express all Ig isotypes but lack secretory IgM due to a targeted mutation of the IgM secretion signal (24). These mice respond to T-independent Ags, are capable of generating levels of total specific IgG comparable to those of wt mice (see also Discussion), and have at least 2-fold more B1b cells than of wt mice (24) (K. R. Alugupalli and J. Chen, unpublished observations). Nevertheless, in sIgM-/- mice infected with DAH-p1, the initial peak of bacteremia was 50-fold higher than that observed after infection of wt mice and persisted for at least 17 days (i.e., throughout the course of the experiment, which was terminated due to severe morbidity; Fig. 6A, left panel). Infection of sIgM-/- mice with the moderate passage strain DAH-p19 resulted in a peak bacteremia >1 order of magnitude higher than that found in wt mice. Even more strikingly, while wt mice cleared strain DAH-p19 after 1 or 2 days, sIgM-/- mice could not clear infection during the entire 3-wk observation period (Fig. 6A, right panel).



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  		FIGURE 6. Secretory IgM is essential for the clearance of B. hermsii. A, 129Sv (sIgM+/+; n = 4) or secretory IgM-deficient (sIgM-/-; n = 6) mice were infected i.v. with 4 x 105 B. hermsii DAH-p1 or DAH-p19, and bacteremia was measured by microscopic counting. The broken line indicates the detection limit for bacteremia. B, Lack of sIgM results in enhanced B. hermsii colonization. Perfused organs of B. hermsii DAH-p1-infected sIgM+/+ (n = 4) or sIgM-/- (n = 6) mice were harvested on the day 20 postinfection, total DNA content was extracted, and the amounts of B. hermsii flaB and mouse nidogen sequences were measured by real-time PCR (see Materials and Methods for details). Bacterial burden was interpreted as the amount of flaB gene copies per 105 nidogen gene copies. The broken line indicates the detection limit. The flaB sequences were undetectable in the perfused organs of wt mice infected with DAH-p1.

 
To test whether the inability of sIgM-/- mice to clear B. hermsii was also reflected in the bacterial burden in multiple tissues, we performed whole body perfusion to minimize blood-derived bacterial contamination and then measured B. hermsii DNA in various tissues by quantitative PCR (see Materials and Methods). At 20 days after infection of wt mice with DAH-p1, B. hermsii DNA could not be detected in any of seven organs, whereas significant bacterial burden was detected in infected sIgM-/- mice (Fig. 6B). Thus, our results demonstrate that IgM is the critical B cell effector molecule that is necessary for the clearance of B. hermsii from both blood and tissue.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To better understand the remarkably rapid clearance of relapsing fever spirochetes from the blood, we sought to identify the critical effector mechanism(s) in a murine model of relapsing fever.

We first demonstrated that SCID and Rag-/- mice were incapable of resolving B. hermsii infection, indicating a critical role of T cells, B cells, or both. TCR-{beta} x -{delta}-/- mice, which lack T cells, and IL-7-/- mice, which are deficient in both T cells and FO B cells, but not in B1 and MZ B cells, cleared B. hermsii efficiently, suggesting that B cell subclasses such as MZ B cells and/or B1 cells are sufficient to eliminate B. hermsii. MZ B cells reside in the spleen, and splenectomized mice demonstrated no defect in bacterial clearance when challenged with a moderate passage strain (i.e., DAH-p19) that grows to 103/µl in blood, indicating that MZ B cells do not play the primary role in eliminating spirochetes. In contrast, xid mice, which are severely deficient in B1 cells, suffered significantly longer and more severe episodes of bacteremia compared with wt mice, suggesting that B1 cells are likely to be the immune cell most critical for the clearance of B. hermsii. This hypothesis was supported by the observation of a remarkable increase in the number of B1 cells in infected xid mice after the eventual resolution of B. hermsii infection. Indeed, an apparent increase in B1 cells in the peritoneum of B. hermsii-infected wt and xid mice was evident by day 7, just after resolution of the first episode of bacteremia, and adoptive transfer of expanded peritoneal B1b cells of wt or xid mice into Rag-/- mice appeared to confer protection from uncontrolled bacteremia (K. R. Alugupalli, unpublished observations). Finally, IgM is the dominant isotype expressed by B1 cells, and mice selectively incapable of secreting IgM were unable to clear B. hermsii.

B1 cells are known to differentiate into Ab-forming cells within 3 days of encountering blood-borne, T-independent Ags (16), which coincides with the kinetics of clearance of B. hermsii from the blood of infected animals. These lymphocytes can enter the cell cycle very rapidly, as B1 cell proliferation in vitro is extensive even 24 h after activation (35). Our results are consistent with the hypothesis that spirochete load, or some factor associated with spirochete load, is an important determinant of the extent of expansion, because a significant increase in peritoneal B1 cells only occurred in mice that suffered high level (e.g., 104/µl) bacteremia; moderate passage strain DAH grew to only 103/µl in the blood and did not trigger a significant expansion of B1 cells in wt mice. Similarly, a moderate dose of heat-killed Streptococcus pneumoniae did not result in the activation of peritoneal B1 cells (22). One caveat to our interpretation of infection by moderate passage B. hermsii is that the genetic lesion(s) responsible for its diminished level of peak bacteremia is not defined and could alter the host immune response by a mechanism(s) independent of bacterial load. Interestingly, although both B1a and B1b cells are known to respond rapidly to antigenic stimulation, B. hermsii infection resulted in a selective expansion of B1b cells. As B1b cells can be generated from the bone marrow by differentiation from progenitor cells and also have self-renewing capability in the peritoneum (17, 36), we cannot yet identify the developmental origin of the expanded population of B1b cells during B. hermsii infection.

A remarkable observation is that B1 cell expansion occurred in xid mice, which are profoundly deficient in B1 cells and B cell Ag receptor (BCR) signaling due to the absence of functional Btk. Indeed, after 4 wk of infection, the number of B1 cells in the peritoneum of xid mice was comparable to that in wt mice. B1 cells from xid mice do not respond well to T-independent type 2 Ags, which activate lymphocytes by cross-linking the BCR (31, 32, 34). Nevertheless, recent evidence indicates that dual engagement of BCR and Toll-like receptors by certain Ags can activate B cells in a T-independent fashion (37). Thus, additional signaling mechanisms may potentiate a weak BCR signal in xid B1 cells. Consistent with such a possibility, B. hermsii membranes are rich in lipoproteins (e.g., Vmp) and such Toll-like receptor ligands may play an essential costimulatory role in B1 cell activation. It will be of interest to identify the precise bacterial component(s) required to trigger the observed expansion of B1 cells.

While we find it likely that B1 cells play the primary role in the clearance of B. hermsii, other immune cells may also contribute to this function. Splenectomized mice, whose peritoneal B1 cell population remains intact, nevertheless demonstrated a significant defect in the clearance of a B. hermsii strain DAH-p1 that produces an extremely high level bacteremia. As splenectomized mice lack MZ B cells, which share several functional similarities with B1 cells, it is tempting to speculate that MZ B cells also contribute to spirochetal elimination under conditions of high bacterial load. Indeed, it has been demonstrated by Kearney and colleagues (22) that both B1 and MZ B cells expand when immunized with S. pneumoniae, which elicits a specific T-independent IgM response. An obvious caveat to our speculation is that the spleen harbors many immune effector cells in addition to MZ cells, including splenic macrophages. In fact, in B cell-deficient mice (13) or SCID or Rag-/- mice (Fig. 1), bacteremia was diminished 10-fold from initial peak levels, suggesting that some innate effector mechanism(s) can partially control relapsing fever bacteremia during the chronic phase.

Presumably, the role of activated B1 and perhaps MZ B cells during B. hermsii infection is to produce the IgM that results in bacterial clearance, because sIgM-/- mice were incapable of eliminating B. hermsii from the blood. Although these mice display a reduced IgG2b response to a model T-independent Ag, they also exhibit an enhanced IgG2a response, resulting in total IgG levels comparable to those in wild-type mice (24). In addition, IgM provides more complete passive protection than IgG2b in a B. duttonii infection model (12), and the production of IgM, not IgG, temporally correlated with clearance of the Spanish relapsing fever spirochete (13). The findings presented here provide genetic evidence of an absolute requirement for sIgM in control of this infection.

B1 cells are the major source of natural IgM, i.e., Abs that occur spontaneously in naive Ag-free mice and in normal individuals in the absence of apparent Ag stimulation (38), and this natural Ab has been shown to play a role in the early control of infection in both bacterial and viral infection models (39, 40, 41). It appears unlikely, however, that natural Abs play the critical role in clearance of B. hermsii; endogenous levels of this Ab are clearly not sufficient to prevent B. hermsii infection in naive wt mice, and transfer of naive serum from normal mice into B cell-deficient (13) or SCID mice (K. R. Alugupalli, unpublished observations) did not confer protection. A serotype-specific IgM mAb partially protected irradiated mice from B. hermsii infection in a serotype-dependent manner (8). Thus, an acquired immune response appears to be required for efficient elimination of B. hermsii, a suggestion consistent with the episodic nature of relapsing fever bacteremia that apparently reflects outgrowth and then elimination of a particular serotype of spirochetes (3). It is noteworthy that B1b cells, which are selectively expanded during B. hermsii infection, have greater Ig junctional diversity than do B1a cells (42) and are therefore perhaps better able to mount a response to a pathogen that undergoes antigenic variation. The specific targets of this putative adaptive B1 cell response as well as the BCR specificity of the responding cells are currently being investigated.

The mechanism by which sIgM promotes clearance of B. hermsii remains unclear. One well-known effector function of IgM is its ability to activate the complement system, but C3 and C5 deficient mice are capable of clearing the Spanish relapsing fever Borrelia as efficiently as wt mice (13). An alternative mechanism is suggested by the finding that Abs or their F(ab)2 have the capacity to destroy Ags through the conversion of molecular oxygen into hydrogen peroxide (43). Interestingly, some mAbs and Fab that recognize surface proteins of the related spirochete Borrelia burgdorferi are bactericidal in vitro in the absence of complement (44, 45). Alternatively, a high affinity IgM receptor, Fc{alpha}/µ, that is capable of promoting endocytosis of IgM-opsonized bacteria in vitro, is highly expressed on the majority of B cells and monocytes in several tissues, including spleen and liver (46), and has been suggested to promote the clearance of relapsing fever (13). Indeed, hepatosplenomegaly is temporally correlated with bacterial clearance in infected mice, and at 2 days postinfection the spirochete burden in the spleen and liver is increased dramatically (K. R. Alugupalli, unpublished observations).

B. hermsii infection of the mouse closely mimics human relapsing fever and has provided an important model for understanding both the mechanisms of antigenic variation and the role this variation plays in immune escape by microbial pathogens (3, 6). In the current study we have developed evidence that a T-independent immune response involving IgM produced by B1b cells promotes the elimination of bacteria from the blood of infected mice. This infection model may provide insights into other infections that depend on T-independent responses for clearance or protection. For example, a T-independent Ab response is required for immunity to the related bacterium, B. burgdorferi, as well as for the resolution of arthritis and carditis in the murine model for Lyme disease (47). Finally, an important limitation of T-independent responses in immunity is the lack of affinity maturation and memory, and the murine model of relapsing fever may promote a better understanding of this class of immune responses and the basis of these limitations.


   Acknowledgments
 
We thank Drs. Richard Murray for providing IL-7-/- mice, Tom Schwan for B. hermsii strain DAH, Drs. Jon Goguen and Jennifer Wilshire for suggestions, and Dr. Janis Weis for helping with the quantitative PCR protocol.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant R01AI 37601 and an American Heart Association Established Investigator award (to J.M.L.), National Institutes of Health Grant R01AI 43534 and a grant from the Charles H. Hood Foundation (to R.M.G.), National Institutes of Health Grant R01AI 41054 (to R.T.W.), and National Institutes of Health Grant R01CA66644 (to E.S.-T.). Back

2 Address correspondence and reprint requests to Dr. Kishore R. Alugupalli, Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: kishore.alugupalli{at}umassmed.edu Back

3 Abbreviations used in this paper: Vmp, variable major protein; BCR, B cell Ag receptor; Btk, Bruton’s tyrosine kinase; FO, follicular; MZ, marginal zone; PerC, peritoneal cells; UMMS, University of Massachusetts Medical School; xid, X-linked immunodeficiency; wt, wild type. Back

Received for publication September 26, 2002. Accepted for publication February 6, 2003.


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 Discussion
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