The Nod2 Agonist Muramyl Dipeptide Cooperates with the TLR4 Agonist Lipopolysaccharide to Enhance IgG2b Production in Mouse B Cells

Many studies have shown that Toll-like receptors (TLRs) and Nod-like receptors (NLRs) were expressed in B cells and their signaling affects B cell functions. Nonetheless, the roles played by these receptors in B cell antibody (Ab) production have not been completely elucidated. In the present study, we examined the effect of the Nod2 agonist muramyl dipeptide (MDP) in combination with the TLR4 agonist lipopolysaccharide (LPS), a well-known B cell mitogen, on B cell viability, proliferation, and activation, and Ab production by in vitro culture of purified mouse spleen resting B cells. MDP combined with LPS to reinforce B cell viability, proliferation, and activation. Moreover, MDP enhanced LPS-induced IgG2b production, germline γ2b transcript (GLTγ2b) expression, and surface IgG2b expression. In an experiment with Nod2- and TLR4-deficient mouse B cells, we observed that the combined effect of MDP and LPS is dependent on Nod2 and TLR4 receptors. Furthermore, the combined effect on B cell viability and IgG2b switching was not observed in Rip2-deficient mouse cells. Collectively, this study suggests that Nod2 signaling enhances TLR4-activated B cell proliferation, IgG2b switching, and IgG2b production.

For instance, TLR2 stimulation arrests TLR4 agonist LPSpromoted B cell maturation [15]; BCR signaling synergizes with TLR signaling for activation-induced cytidine deaminase (AID) expression and Ig class switch recombination (CSR) by B cells [16]. Thus, TLRs play various roles in B cell activation, differentiation, and function.
However, the roles played by NLRs (Nod1, Nod2, NLRC4, NLRP3, etc.) in B cells remain to be elucidated. The effects of Nod1 and Nod2 stimulation on B cell activation have been investigated in only a few studies: Cohen and Parant reported that Nod2 agonist muramyl dipeptide (MDP) increases surface Ig (membrane κ-light chain) expression and enhances the response to LPS in the mouse pre-B cell line 70Z/3 [17]; Petterson et al. reported that Nod1 or Nod2 stimulation augments BCR-or TLRinduced human B cell activation (proliferation, viability, and expression of cell surface markers) independently of physical T cell assistance [18,19].
In the present study, to further elucidate the role of Nod2 in B cell response, we directly stimulated mouse resting B cells with MDP in the absence and presence of LPS in vitro and analyzed B cell viability, proliferation, activation, antibody (Ab) production, and Ig class switching.
2.3. Cell Viability, Proliferation, and Activation Assays. Cell viability was determined using the EZ-Cytox cell viability assay (DaeilLab Service Co., Ltd., Seoul, Korea) according to the manufacturer's instructions [8]. For the cell proliferation assay, purified mouse resting B cells were labeled with CFSE (eBioscience) and then supplemented with MDP, iE-DAP, and LPS. CFSE dilution was measured by counting 10,000 cells with the FACSCalibur. For the cell activation assay, cultured cells were stained with anti-CD69-FITC (BD Biosciences), and the expression levels were analyzed by flow cytometry (FACSCalibur).

Isotype-Specific ELISAs.
Antibodies produced in B cell cultures were detected using isotype-specific ELISAs as previously described [8].
2.5. RNA Isolation and RT-PCR. RNA isolation and RT-PCR were performed as previously described [6]. The PCR primers (Supplementary Table 1) were synthesized by Bioneer (Daejeon, Korea). PCR for β-actin was performed in parallel to normalize for cDNA concentrations within each set of samples. PCR products were resolved by electrophoresis on 2% agarose gels. Semiquantitative RT-PCR analysis was performed using cDNA dilutions.   or anti-mouse IgM-PE in the dark for 30 min at 4°C, and surface Ig-expressing B cells were analyzed by flow cytometry (FACSCalibur). Dead cells were excluded from analysis using Zombie Red™ Fixable Viability Kit according to the manufacturer's instruction (BioLegend, San Diego, CA).

Statistical Analysis.
Statistical differences between experimental groups were determined by analysis of variances. All p values were calculated using unpaired 2-tailed Student's t -tests to assess statistical significance.  (Figure 1(b)). However, MDP alone did not induce any Ab production (data not shown). These results suggest that MDP sustains B cell viability, but MDP itself hardly induces B cell proliferation and plasma cell differentiation.

Nod2
Agonist MDP but Not Nod1 Agonist iE-DAP Combines with TLR4 Agonist LPS to Induce B Cell Viability and Proliferation and IgG2b Production. New functions in innate immune cells have been reported for the crosstalk between TLRs and NLRs [20][21][22]. There is a synergistic stimulation of human monocytes and dendritic cells by TLR4 and Nod1-and Nod2-activating agonists [23]. Furthermore, Nod2 is involved in TLR4-mediated signaling of inflammation regulation [24,25]. LPS stimulates TLR4 and is a well-known mitogen for mouse B cells [26,27]. TLR4 on B cells recognizes LPS and stimulates B cell proliferation, differentiation, and Ig CSR. LPS in vitro stimulation increases IgG2b and IgG3 production through IgG2b and IgG3 class switching, respectively, by mouse B cells [4,[28][29][30][31][32][33]. Therefore, we investigated the combined effect of TLR4 agonist LPS and Nod2 agonist MDP or Nod1 agonist iE-DAP on B cell viability, proliferation, activation, and Ab production. Resting B cells were stimulated with MDP or iE-DAP in the presence or absence of LPS. After 2 and 3 days of culture, LPS-induced cell viability was significantly enhanced by MDP but not by iE-DAP (Figure 2(a)). In addition, MDP reinforced LPS-induced cell proliferation (Figure 2(b)). These results indicate that MDP combined with LPS to induce B cell viability and proliferation, while iE-DAP does not. Further, MDP enhanced LPS-induced expression of CD69, which is an activation marker (Figure 2(c)). Next, we examined the effect of MDP on LPS-induced Ab production, particularly IgG2b and IgG3 production. MDP increased LPS-induced IgG2b production but decreased LPS-induced IgG3 and IgG1 production    ( Figure 2(d)). iE-DAP had no significant effect on LPSinduced Ab production. Instead, iE-DAP decreased LPSinduced IgG2b production. These results indicate that MDP combines with LPS to selectively induce IgG2b production.

MDP Combines with LPS to Induce Germline γ2b
Transcripts and Surface IgG2b Expression. The transcription of germline transcripts (GLT) is a prerequisite for subsequent Ig CSR [34][35][36]. Therefore, GLT expression can serve as a marker of Ig class switching. LPS induces the expression of GLTγ2b as well as that of GLTγ3 [34,37,38]. To evaluate the effect of MDP on LPS-induced IgG2b class switching, we examined whether LPS and MDP together induce the expression of germline γ2b transcripts (GLTγ2b) and surface IgG2b. Resting B cells were stimulated with MDP and iE-DAP in the presence or absence of LPS, and GLT expression were measured by RT-PCR (Figure 3). LPS-induced GLTγ2b expression was enhanced by MDP, whereas MDP did not affect LPSinduced GLTγ3 and GLTγ1 expression (Figure 3). In contrast, iE-DAP neither had any effect on LPS-induced GLTγ2b expression nor on GLTγ3 and GLTγ1 expression. Because AID is an essential enzyme for class switching [39], we assessed its expression. MDP did not affect LPSinduced AID mRNA expression (Figure 3). In addition, MDP selectively enhanced LPS-induced surface IgG2b expression (Figure 4). MDP alone did not induce surface IgG2b expression (data not shown). Collectively, these indicates that MDP can enhance LPS-induced B cell proliferation through Nod2. Next, we examined the effects of LPS and MDP on Ab production in TLR4-and Nod2-deficient B cells. TLR4-deficient B cells did not produce all Abs production upon stimulation of LPS ( Figure 5(b)). In Nod2-deficient B cells, MDP did not increase LPS-induced IgG2b production. Furthermore, MDP did not increase LPS-induced GLTγ2b expression in Nod2-deficient B cells ( Figure 5(c)). These results suggest that the combined effect of LPS and MDP on B cell proliferation and IgG2b production is dependent on their receptors, TLR4 and Nod2. In addition, we investigated the effects of LPS and MDP on B cell responses in receptor-interacting protein 2 (Rip2)-deficient (Rip2 -/-) B cells (Figure 6), because Rip2 is a critical mediator of Nod2 signaling in innate and adaptive immune responses [40][41][42][43]. MDP neither reinforced LPS-induced cell viability nor increased cell proliferation (Figure 6(a)), IgG2b production ( Figure 6(b)), or GLTγ2b expression ( Figure 6(c)) in Rip2-deficient B cells. Thus, Nod2-Rip2mediated signaling could cooperatively play a critical role in LPS-induced B cell responses. However, the underlying molecular mechanisms remain to be determined.

Conclusions
Our present observations demonstrate that direct stimulation of Nod2 selectively enhances TLR4 agonist LPS-induced IgG2b production by enhancing IgG2b class switching in mouse B cells. IgG2b is particularly important early in the immune response, when T cell support may be limited (i.e., T-independent response), and provides early FcγR-mediated effector functions and efficient complement activation through binding on C1q [31,[44][45][46]. Consequently, Nod2 agonist MDP can be used as B cell adjuvant to protect from fast-replicating bacterial infection through enhancing direct B cell activation and IgG2b production independent of T cells and BCR stimulation.

Data Availability
All data supporting the findings of this study, including its supplementary information files, are available from the corresponding author upon reasonable request.

Disclosure
The preliminary results of the current work have been presented as poster presentation on the 15 th International Congress of Immunology 2013 (Milan, Italy; Abstract no.: P3.07.27). Lee Sang-Hoon's present address is the Curocell Inc., Daejeon, Republic of Korea.

Conflicts of Interest
The authors declare no financial or commercial conflict of interest.