Immunoglobulin M (IgM) exists as two types: natural IgM and immune IgM (1). Natural antibodies, consisting mostly of the natural IgM, are the antibodies found in the sera of humans and mice without any exposure to antigens (2). In contrast to the natural IgM, immune IgM is the first antibody produced in response to antigenic stimulation (3). Both IgMs have an important role in pathogen defense, especially bacterial infection (4). Several investigators have suggested that the administration of IgM in both
DNA containing CpG dinucleotide motifs (CpG-DNA), which are found in bacterial DNA, stimulate Toll-like receptor 9 (TLR9) on immune cells, leading to cell proliferation and cytokine production (12). Furthermore, CpG-DNA activates B cells, producing T cell-independent antibodies (13). These roles of CpG-DNA in the immune system are closely related to the prevention of bacterial infection (14–17). Investigators have reported that CpG-DNA activated dendritic cells and the secreted type-1 cytokine, IFN-γ, have protective roles against bacterial infection in murine models (14, 15). The antibacterial effects of reactive oxygen species (ROS) produced by the administration of CpG-DNA in osteoblast-like cell lines have also been shown in
Previously, we reported that bacteria-reactive IgG produced by the administration of CpG-DNA enhances the antibacterial effect in murine models (18); however, the biological functions of IgM produced by CpG-DNA administration are not known. Here, we confirmed that the production of TLR9-mediated IgM occurred both in the peritoneal cavity and sera of mice and that IgM was produced in the peritoneal B cells
In our previous studies (18), we confirmed that the survival of mice after
To analyze the binding ability of IgM produced by the CpG-DNA administration to various bacterial species, we selected Gram-positive bacterial species (
To determine whether the IgM production induced by the CpG-DNA administration was due to the activation of the TLR9 signaling pathway, we performed the same experiments using BALB/c TLR9−/− mice. There were no significant changes in IgM production both in the peritoneal fluid and sera of the TLR9−/− mice even with the injection of CpG-DNA 1826 (Fig. 1G–J). Therefore, the results suggest that the TLR9 signaling pathway is involved in the production of bacteria-reactive IgM induced by CpG-DNA stimulation.
To identify where the bacteria-reactive IgM was produced by the CpG-DNA administration, the mice were intraperitoneally injected with CpG-DNA 1826 or non-CpG-DNA 2041, and then the peritoneal fluid, spleen, mesenteric lymph nodes (MLN), and cervical lymph nodes (CLN) were harvested from the mice 7 days after the injection. The cells from the tissues were cultured for 2 days, and the levels of IgM were analyzed in the cell culture supernatants (Fig. 2A). The IgM amounts were increased in the culture supernatants of cells prepared from the CpG-DNA 1826-injected peritoneal fluid, but were not increased in the other tissues by CpG-DNA 1826 injection. To determine whether IgM is produced by CpG-DNA stimulation
To obtain the bacteria-reactive monoclonal IgM antibody from the mouse peritoneal cavity cells, we screened the hybridoma cells secreting CpG-DNA-induced IgM. Seven days after the intraperitoneal injection of CpG-DNA 1826, the peritoneal cavity cells were harvested from the mice, and they were fused with myeloma SP2/0 cells, and screened in selective medium. After screening a hybridoma clone (3F3F5) secreting the bacteria-reactive monoclonal IgM antibody, the cells were cultured serum-free medium for 5 days. The bacteria-reactive monoclonal IgM antibody was purified from the culture supernatants using ion-exchange chromatography, followed by gel filtration chromatography. The IgM protein fractions were collected, loaded and run on a SDS-PAGE, and stained with Coomassie brilliant blue R solution (Fig. 3A). The fractions including the purified IgM were analyzed by Western blotting (Fig. 3B). We confirmed the ability of the purified monoclonal IgM antibody to bind several Gram-positive bacteria by ELISA (Fig. 3C).
To perform the phagocytosis assay
In our previous experiments, we confirmed that CpG-DNA treatment enhanced the host protection following
Natural antibodies, which exist without any exposure to antigens, have a broad antibacterial activity, and prevent several bacterial infections (24). Here, we confirmed that the amount of IgM produced by the CpG-DNA treatment was greatly increased in the peritoneal cavity of the mice and had a broad reactivity against several bacteria (Fig. 1). We suppose that the bacteria-reactive IgM has broad reactivity because the epitopes of the antibody might be common bacterial surface components such as carbohydrate, glycoprotein, and/or metabolites.
In addition, the secretion of IgM antibodies by the CpG-DNA treatment was significantly increased in the peritoneal cells when we isolated the peritoneal cells, spleen, MLN, and CLN and stimulated them with CpG-DNA
Researchers have shown the efficacy of antibody-mediated phagocytosis against bacterial infection as a measure of the anti-bacterial effect in phagocytic cells (24, 27). When we examined the effects of the bacteria-reactive monoclonal IgM antibody (3F3F5 mIgM) against bacterial infection, the efficacy of phagocytosis against
In conclusion, we proved that the bacteria-reactive IgM produced by CpG-DNA has protective roles against
CpG-DNA 1826 and non-CpG-DNA 2041, whose backbones of the sequences were modified with phosphorothioate, were synthesized by GenoTech Co. The following sequences were used: CpG-DNA 1826, 5′-TCCATGACGTTCCTGACGTT-3′ and non-CpG-DNA 2041 as a negative control, 5′-CTGGTCTTTCTGGTTTTTTTCTGG-3′. The oligodeoxynucleotides were dissolved in distilled water, injected intraperitoneally in BALB/c mice or TLR9−/− mice (50 μg/mouse).
Primary cells were harvested from the mice, homogenized, and re-suspended in RPMI 1640 medium containing 5% FBS. After removing the erythrocytes, the cells were cultured in RPMI 1640 medium containing 5% FBS with 100 U/ml of penicillin and 100 μg/ml of streptomycin. Next, each cell culture plate was treated with PBS or 5 μg/ml of CpG-DNA 1826 or 5 μg/ml of non-CpG-DNA 2041. After 48 h, the cell culture supernatants were harvested and analyzed by ELISA to quantify the IgM levels.
Peritoneal cells were stained with antibodies in sorting buffer (1 mM EDTA, 25 mM HEPES pH 7.0, 1% FBS diluted in PBS). To isolate the B cells, the peritoneal cells were incubated with anti-mouse CD19 (BD Biosciences, San Jose, CA, USA) and anti-mouse CD23 (eBioscience, San Diego, CA, USA). To sort non-B cells from the peritoneal cells, anti-mouse CD3 (BD Bioscience) was used to isolate T cells. B1 cells and B2 cells were sorted using a FACSAria™ II system (Becton Dickinson Inc.).
IgM-secreting hybridoma cells (3F3F5 clone) were applied to the Hybridoma-SFM (Thermo Fisher Scientific Inc.), and grown at 125 rpm in a shaker incubator containing 8% CO2 at 37°C. After 5 days, cell culture supernatants were obtained by centrifugation at 3,000 rpm for 10 min, and purified using DEAE Sepharose™ Fast Flow (GE Healthcare Co.) and HiPrep™ 16/60 Sephacryl™ S-300 HR (Ge Healthcare Co.). The samples were loaded onto a SDS-PAGE, analyzed by Coomassie blue staining and Western blotting (28).
The purified IgM was resolved, and transferred onto nitrocellulose membranes, which were blocked with 5% Skim milk in PBS-T for 1 h at room temperature. Membranes were incubated with HRP-conjugated goat anti-mouse IgM (μ-chain specific) antibody (Merck Millipore Co.) for 2 h at room temperature. Immuno-reactive protein band intensities were measured with chemiluminescence reagent (Thermo Fisher Scientific Co.) as previously described (29).
The mouse RAW 264.7 macrophage cell line was purchased from the American Type Culture Collection (ATCC). The cells were cultured overnight on poly-L-lysine-coated cover glass (Sigma-Aldrich Co.) in 12-well plates (Nunc Inc.). The FITC-labeled
Additional Material and Methods are provided in the Supplementary Material.
This work was supported by grants from the National Research Foundation (2017R1A2B2007373, 2009-0093812) funded by the Ministry of Science and ICT in the Republic of Korea.
The authors have no conflicting interests.
Production of bacteria-reactive IgM in the mouse peritoneal cavity and serum by administration of CpG-DNA 1826. (A, B) BALB/c mice were intraperitoneally injected with CpG-DNA 1826. After 7 days, the mice were intravenously injected with
Production of bacteria-reactive IgM in the mouse peritoneal cavity cells by CpG-DNA 1826 treatment
Purification and characterization of monoclonal IgM antibody produced by CpG-DNA 1826 stimulation. (A, B) IgM-secreting hybridoma cells (clone 3F3F5) were cultured in Hybridoma-SFM for 5 days. The supernatants of the cell cultures were collected, and subjected to DEAE-sepharose column chromatography, and then, the protein fractions containing IgM were purified by gel filtration. The fractions containing monoclonal IgM antibody were analyzed by Coomassie brilliant blue staining (A) and Western blotting (B). S, cell culture supernatants; HC, heavy chain; LC, light chain. (C) The bacteria-reactivity of the purified IgM was measured by ELISA using plates coated with Gram-positive bacteria (n = 3/group). The results presented are representative of three experiments.
Enhanced phagocytosis activity in RAW 264.7 cells by the bacteria-reactive monoclonal IgM antibody. FITC-labeled