In Situ IgM Production and Clonal Expansion of B-1 Cells in Peritoneal Cavity Promote Elimination of C. albicans Infection in IgH Transgenic Mice with VH Derived from a Natural Antibody

B-1 cells are innate-like cells that play important roles in host defense against infection. However, the function of B-1 cells in fungi infection remains unclear. Previously we produced IgH transgenic mice TgVH3B4 with VH derived from a natural antibody 3B4 that can identify C. albicans, and found that TgVH3B4 mice were resistant to intraperitoneal (i. p.) and intravenous C. albicans infection. Most of the peritoneal cavity (PEC) B-1 cells in TgVH3B4 mice express transgenic BCR that binds C. albicans. In the present study, we explored the response of B-1 cells to C. albicans infection by applying i. p. inoculation of fungi in TgVH3B4 mice. We found that C. albicans was cleared more efficiently in TgVH3B4 mice after i. p. inoculation than that of littermate control. The level of C. albicans-reactive IgM in PEC of TgVH3B4 mice was much higher than that of control, and the number of B-1a B cells was also elevated in TgVH3B4 mice, which was mainly due to enhanced proliferation of B-1 cells. Additionally, numbers of C. albicans-specific B cells increased greatly in TgVH3B4 mice after C. albicans inoculation. Our data suggested that in situ IgM production and clonal expansion of B-1 cells in PEC participate in host defense against C. albicans infection.


Introduction
Natural antibodies, major component of the innate humoral immunity [1,2], have been shown to be important in innate and adaptive immune responses against microbial infections [3][4][5][6][7]. The poly-reactive natural IgM and surface antigen receptor expressed by B-1 cells might be a kind of pattern recognition receptors [8], and can recognize invariant molecular structures shared by large groups of pathogens, including many fungi [8][9][10]. The protective role of natural antibodies against bacteria and virus infection has long been recognized [4,6], however, the importance of natural antibodies in host defense against fungi infection has not been revealed untill recently. B cell-depleted mice have been shown to be more susceptible to systemic candidiasis than controls [11], and B cell knockout mice are easier to develop systemic, but not mucosal, candidiasis [12]. More recently, Kozel's group showed that a mannan-specific IgG antibody from normal human mediates C3-binding and killing of C. albicans [13,14]. We produced a monoclonal polyreactive natural antibody [8,15], 3B4, which not only recognizes self-antigen keratin, but also recognizes a surface antigen located in the germ tubes of C. albicans, and protects mice from C. albicans-induced death with passive immunization, by mechanisms involving suppression of germ tube formation and modulation of phagocytosis [8]. Using the V H gene from 3B4, we constructed a m chain transgenic mouse (TgV H 3B4) which has elevated serum anti-C. albicans IgM and is resistant to C. albicans infections. Analyses of B cell development showed that in TgV H 3B4, most B cells secreting C. albicans-reactive antibodies are selected into the B-1 B cell compartment [8]. These results demonstrate a key role of natural antibodies in anti-C. albicans immune responses, and also indicate that B-1 cells may be important in anti-fungi immunity.
B-1 cells are distinguished from other subsets of B cells by their anatomical localization, phenotype, self-renewing capacity, and production of natural antibodies [16,17]. B-1 cells constitute a major fraction of B cells present in peritoneal cavity (PEC), and are a minor fraction of B cells in spleen [16,17]. Based on cell surface CD5 expression, B-1 cells are subdivided into the B-1a (CD5 + ) and B-1b (CD5 2 ) subsets, and B-1 cells in PEC also express Mac-1. Although the origin of B-1 cells is still in debate, there is evidence indicating that B-1 cells are positively selected by self-antigens, and strong BCR antigen signals appear to be important for the decision to become B-1 cells [16,18,19]. B-1 cells are characterized by the production of natural antibodies in the absence of apparent infection or immunization [16], and B-1-specific antibodies are involved in many biological events, such as reducing atheroscle-rotic lesions, activating T cell responses, contributing to autoimmunity, and promoting ischemia/reperfusion injury [20][21][22]. Most importantly, B-1 cells are envisioned as key players in the early humoral response against pathogens and are thought to be the primary antibody producers in response to T cell-independent type 2 (TI-2) antigens along with marginal zone B cells [23][24][25]. However, the role of B-1 cells in fungal infection and how B-1 cells respond to pathogen infection in peritoneal cavity are still not clear.
In the present study, intraperitoneal (i. p.) C. albicans inoculation was applied to our transgenic model of TgV H 3B4, in which C. albicans-reactive B-1 cells were enriched in PEC, to explore the role and immune response of B-1 cells in fungi infection. Our data showed that C. albicans-specific B-1 cells proliferated actively and produced a large amount of natural IgM to clear the pathogen, indicating that B-1 cells play a critical role in host defense against C. albicans infection.

Results
Efficient clearance of C. albicans in PEC of TgVH3B4 In previous study, we found TgV H 3B4 mice were resistant to both intravenous (i. v.) and i. p. C. albicans infection. In particular, almost complete protection was observed in i. p. inoculation with a relatively high number of fungi in TgV H 3B4 mice [8]. It seemed likely that local environment of PEC might exert more efficient defense against C. albicans infection. In this study, we set out to analyze in detail the antibody and B-1 cell responses in PEC after i. p. C. albicans inoculation. A lower dose of C. albicans (2610 6 ) was applied for i. p. inoculation, and our previous study has shown that the infection would be restricted in PEC and all the mice would survive.
The C. albicans burden and inflammatory infiltration in PEC after fungi inoculation were analyzed in TgV H 3B4 mice and littermate control. At different time point after C. albicans inoculation, the lavages were eluted from the mice. The supernatant of the elution was subjected to inflammatory cytokine analysis, and the cells in the elution were subjected for colonyforming assay and FCM analysis. The colony-forming units (CFU) of the elution after C. albicans inoculation decreased with time, and level of inflammatory cytokines and neutrophil infiltration increased gradually and began to decrease from 36h after inoculation. The data at 36 h after inoculation were shown in Fig. 1. The number of living yeasts in the lavage of TgV H 3B4 mice was much lower than that of control (Fig. 1A). The concentrations of inflammatory cytokines, TNF-a, IL-6 and MCP-1, were approximately 3 folds lower in TgV H 3B4 mice than that of their littermates (Fig. 1B). There were fewer neutrophils in the PEC lavage of TgV H 3B4 mice when analyzed using anti-Gr1 antibody (Fig. 1C). These data showed that there were less C. albicans burden, less neutrophil infiltration and inflammatory cytokines in PEC of TgV H 3B4 mice, indicating that C. albicans was cleared more efficiently in TgV H 3B4 mice.

Elevated IgM production in PEC of TgVH3B4
B-1 cells are the major source of natural IgM antibodies which constitute the first line of defense against infection. In the TgV H 3B4 mice, most B cells in PEC produce the 3B4 IgM that can recognize C. albicans [8,26]. Our previous study have demonstrated that the 3B4 antibody protected mice from C. albicans-induced death in passive immunization, by mechanisms involving suppressing the germ tube formation and modulating phagocytosis. Thus, to elucidate the mechanism for the enhanced clearance of C. albicans in TgV H 3B4 mice, we first analyzed the antibodies production in PEC before and after fungi inoculation.
Before fungi inoculation the total IgM level was almost the same between TgV H 3B4 mice and control, however, the level of C. albicans-specific IgM in PEC of TgV H 3B4 mice was much higher than that of control ( Fig. 2A-B). After C. albicans inoculation, the total IgM level increased slightly in both TgV H 3B4 and control mice, as shown in Fig. 2A. However, the level of C. albicans-specific IgM increased greatly in TgV H 3B4 mice, in contrast to a slight increase of anti-C. albicans IgM in the control mice (Fig. 2B). There was no difference of total and C. albicans-specific IgG antibodies in PEC elution before and after inoculation between TgV H 3B4 mice and control (data not shown). These data indicated that B-1 cells produce natural IgM in PEC at a baseline level in normal conditions, and are stimulated to produce larger amount of antigen-specific antibodies upon pathogen inoculation.

Increased proliferation of B-1 cells after C. albicans inoculation
As B-1 cells are the major producing cells of natural antibodies, we analyzed B-1 cells' number and proliferation in PEC after C. albicans infection. Dynamic analysis of B cell numbers showed that there was a transient decrease of B-1 cell numbers in PEC after C. albicans inoculation, and then the numbers increased and peaked at 36h after inoculation (data not shown). The subsets of B-1 cells were analyzed at 36 h after inoculation as shown in Fig. 3. The number of CD5 + B-1a cells in TgV H 3B4 was higher than that of littermate before C. albicans inoculation (Fig. 3), which has been reported in our previous study [8]. While after C. albicans inoculation, numbers of B-1a cells increased in both TgV H 3B4 and littermate, and TgV H 3B4 has higher number of B-1a cells.
The increase in B-1a cell number may be the result of enhanced proliferation or accelerated migration. Consequently, we further assessed the proliferation of B-1 cells by using BrdU incorporation assay. Results showed that before PEC inoculation there was only a detectable incorporation of BrdU in B-1a and B-1b cells in both TgV H 3B4 and control mice (data not shown). After PEC C. albicans inoculation, there were marked proliferation of both B-1a and B-1b cells, as shown by 19% and 10% BrdU + cells (Fig. 4). The proliferation of B-1a cells in TgV H 3B4 was greater than that of B-1b cells, indicating that B-1a cells responded more actively in immediate immunity to C. albicans infection.

Clonal expansion of C. albicans-specific B cells in TgVH3B4
To determine the antigen-specificity of the proliferating B cells after C. albicans inoculation in PEC, we sorted B cells by magnetic beads from PEC, and analyzed by using ELISPOT assay with keratin as coating antigen [8], as polyreactive natural antibody 3B4 can identify both C. albicans and keratin. Before C. albicans inoculation, there were more keratin reactive B cells in PEC of TgV H 3B4 mice than control, which has been shown in previous study [8,26]. After i. p. C. albicans inoculation, there were even more B cells secreting keratin-reactive IgM in TgV H 3B4 mice (Fig. 5A). In the PEC of littermate control, there were also more keratin-reactive B cells after fungal inoculation, but the increasing scope was very limited (Fig. 5A). When B cells from PEC were cocultured with irradiated C. albicans, both total IgM and C. albicansspecific IgM in the supernatant were much higher in TgV H 3B4 mice than that of control (Fig. 5B). These data indicated that there was active clonal expansion of C. albicans-specific B-1 cells in TgV H 3B4 mice after infection with C. albicans.

Discussion
B-1 cells reside predominantly in the PEC, bearing several features of activated B cells, and act as key players in the early humoral response against pathogens by producing natural antibodies [16]. From an evolutionary point of view, it is interesting to note that B-1 cells may represent a transitional phase on the evolution of the adaptive immune response, guarding the main compartments of the primitive organism, the body cavity [27]. One function of peritoneal B1 cells is to survey the abdominal cavity and to join forces with innate cells, such as    macrophages, for a rapid and efficient pathogen clearance. C. albicans is the main commensal organism of the gastrointestinal tract, and it is reasonable to speculate that B-1 cells might have a role against C. albicans infection. In the past several years, it has been reported that B-1 cells play an important role in host defense against many pathogens including bacteria [4,23], viruses [6] and lymphatic filarial parasites [28]. However, the role of B-1 cells in fungi infection remains unclear. Using a monoclonal antibody 3B4 and TgV H 3B4 mice with V H derived from 3B4, we have previously demonstrated a key role of natural antibodies in immune responses against C. albicans infection [8]. In this study, we explored the role and responses of B-1 cells to C. albicans infection by using i. p. fungi inoculation. We found that TgV H 3B4 mice cleared the peritoneal C. albicans infection more efficiently, and level of C. albicans-reactive IgM in the PEC of TgV H 3B4 mice was higher than that of control. Number of B-1a cells was also increased, which was mainly due to enhanced B-1 cell proliferation. Furthermore, C. albicans-specific B cells increased numerously in TgV H 3B4 mice after C. albicans infection. These data suggested that upon peritoneal C. albicans infection, C. albicansspecific B-1 cells proliferate actively and produce a large amount of natural IgM to clear the pathogen. It also indicated that B-1 cells play a critical role in host defense against fungal infection.
One of the most important functions of B-1 cells is producing natural antibodies; however, where B-1 cells produce natural antibodies is not fully understood. Studies showed that PEC B cells are precursors of splenic IgM natural antibody-producing cells and IgA producing plasma cells in murine gut [29,30]. Recently, Fagarasan et al found that antibody production by B-1 cells is closely related with their migration capacity to other lymphoid organs [31]; Wen et al showed that B-1 cells migrate to spleen and produce antibodies [32]. In our study, however, there was anti-C. albicans natural antibodies exist in the PEC of TgV H 3B4 and littermate, and TgV H 3B4 mice had much higher level of C. albicans-specific natural antibodies, indicating that B-1 cells can also produce natural antibodies in PEC without the need for migration.
After C. albicans inoculation in PEC, there was a rapid increase of C. albicans-specific IgM and C. albicans-specific B cells, indicating that the pre-existing C. albicans-reactive B-1 cells in PEC acted actively in early defense against infection. Clonal expansion of B-1 cells after infection has been discussed in several literatures; however, the data from different studies are inconsistent with one other. Baumgarth et al found that the protection against influenza virus infection by B-1 cells-derived IgM antibodies does not entail significant increases in their normal serum levels after infection [33], suggesting that clonal expansion and increased IgM secretion is not initiated in vivo after infection. In Matin's paper, however, T15 idiotype expressing B-1 cells expanded vigorously in both PEC and spleen [25]. Our results are in consistent with Martin's study: C. albicans-specific B-1 cells proliferate in the PEC, and produce a high amount of natural antibody to clear C. albicans infection. The absence of B-1 cell expansion in Baumgarth's work may be due to a different route of infection: blood steam transmitted virus may not have the chance to meet B-1 cells in the spleen and PEC. While in our and Matin's experiment, i. p. infection were applied, and B-1 cells could contact with the pathogen and were stimulated to proliferate and produce antibody. Besides natural antibodies and B-1 cells, there are many other components of innate immunity against C. albicans infection in PEC, including epithelial cells, macrophages, neutrophils, NK cells, complement, and so on. Mechanisms of cooperation between different cells remain to be further investigated in the future.
As the transgene used to construct the TgV H 3B4 mice contains only the IgH m chain; consequently, the effect of other isotypes of immunoglobulin such as IgG cannot be explored using our system. The infection in our model was limited in the PEC, and the role of B-1 cells in disseminated C. albicans infection cannot be explored. More studies are needed to further our understanding on the role of B-1 cells in host defense against fungi infection.

Ethics Statements
The animal husbandry, experiments and welfare were conducted in accordance with the Detailed Rules for the Administration of Animal Experiments for Medical Research Purposes issued by the Ministry of Health of China, and were approved by the Animal Experiment Administration Committee of Fourth Military Medical University. Mice were raised in the specific pathogen free conditions on the C57BL/6 background, and were manipulated with every specific care to reduce the suffering of the mice during the experiments. Mice Transgenic mice with V H derived from a monoclonal antibody 3B4 were generated by our group as described previously [8]. The mice were housed in special pathogen free conditions, and backcrossed with C57BL/6 mice for more than eight generations before analysis. All animal protocols were approved by the University's Animal Research Committee.

Fungal inoculation and PEC lavage preparation
C. albicans (ATCC 90028) were cultured in the Sabouraud's dextrose medium. For in vivo inoculation, 2610 6 C. albicans yeasts were injected i. p. into 6,8-week-old TgV H 3B4 mice and littermate control. At different time point after inoculation (6 h, 12 h, 24 h, 36 h) the mice were sacrificed. PEC lavage was eluted as described previously. In brief, a small hole was cut by scissors on the abdomen wall of the mice, and 1 ml PBS was injected into the PEC with a pipette. Then a pasture tube was used to collect the cell suspension from the PEC. The same procedure was repeated three times and about 3.5 ml lavage from the PEC can be harvested. The supernatant of the first 1 ml of elution was saved for cytokine analysis, and the cells from all the three elution were pooled and subjected to flow cytometry (FCM) analysis and colony-forming assay immediately. Three mice were included in one group and each experiment was repeated for at least three times.

Colony-forming assay and cytokine detection
Living yeasts were revealed by a colony-forming assay. In brief, the elution were spread onto an agar plate and cultured at 37uC overnight, and then the colonies were counted. Cytokines in the elution were analyzed by using a Cytometry Beads Array kit (BD Bioscience, San Diego, CA) according to the manufactures instruction.

ELISA
To measure C. albicans-reactive IgM, C. albicans yeasts were seeded in microtitre plates (Nunc, Roskilde, Denmark) at a density of 1610 5 /100 ml in RPMI 1640 and cultured at 37uC for 2 h to let yeast cells adhere tightly onto the plates, as described previously [8]. For analysis of mouse IgM, goat anti-mouse m chain antibody (Sigma-Aldrich) was coated to microtitre plates at a concentration of 1 mg/ml. PEC lavage from C. albicans inoculated TgV H 3B4 and control mice or supernatant of cell culture were added and incubated at room temperature for 1 h. After washing, the biotinlabeled anti-IgM (Sigma-Aldrich) was added and incubated, followed by incubation with the streptavidin-horseradish peroxidase, and developed by O-phenylenediamine.

BrdU incorporation
0.6mg (200 mL/mouse) BrdU (5-Bromo-2-deoxyuridine) (Sigma-Aldrich) was i. p. injected with fungal inoculation to TgV H 3B4 mice and littermate control, and was injected for the second time 12 h later. After 36 h of inoculation, cells from PEC of mice were subjected for BrdU staining, which was performed using a BrdU Labeling kit (BD PharMingen, San Diego, CA) according to the manufacturer's instructions. Cells were then analyzed by FCM.

Enzyme-linked immunoSPOT (ELISPOT) assay
PEC B cells were sorted by first staining with anti-mouse CD19biotin (BD Biosciences) and then by BD IMag magnetic beads conjugated with avidin (BD Biosciences) according to the manufacturer's instructions. Purified B cells were plated at 2610 5 cells/well in 96-well plates pre-coated with 20 mg/ml of purified mouse epidermis keratin, as the 3B4 antibody can also bind keratin. Cells were stimulated with LPS (50 mg/ml, Sigma-Aldrich) for 5 days at 37uC in an atmosphere of 5% CO 2 . Wells were washed thoroughly with PBS containing 0.02% Tween-20, and the biotin-labeled anti-IgM was added. Plates were incubated for 2 h at room temperature, washed, and incubated with the streptavidin-alkaline phosphotase (Jackson Immunoresearch, Boston, MA) for 75 min at room temperature. Spots were developed by addition of the BCIP substrate (Sigma-Aldrich), and the reaction was stopped by washing extensively with water. Wells were counted under a microscope.
In vitro cell culture PEC B cells were sorted as described above. The sorted cells were plated at a density of 1610 5 cells/well in 96-well plates. cirradiated C. albicans yeasts were seeded in the well at 5610 5 cells/ well and co-cultured with PEC B cells. After 3 days, the supernatant of culture was collected and subjected to ELSA analysis of total IgM and C. albicans-specific IgM.

Statistics
ANOVA was used to determine the statistical significance of values among experimental groups. Statistical significance was defined as p,0.05.