Leuconostoc citreum HJ-P4 (KACC 91035) regulates immunoglobulin E in an ovalbumin-induced allergy model and induces interleukin-12 through nuclear factor-kappa B and p38/c-Jun N-terminal kinases signaling in macrophages

Animals

Male Balb/c mice and C57BL/6 mice at 8 weeks of age were purchased from Charles River Korea (Orient, Korea) and maintained with rodent chow and water ad libitum in a temperature- and humidity-controlled pathogen-free animal facility at Kyunghee University. Mice were cared for according to the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, USA). All experiments were approved by our institutional ethical committee for animal welfare.

Preparation of microorganisms

Leuconostoc citreum HJ-P4 (KACC 91035) was provided by Professor Nam Soo Han at Chungbuk National University. L. citreum was cultured in MRS broth (DIFCO, Detroit, MI, USA) for 16 hr at 30°C, collected by centrifugation, washed several times with sterile distilled water and lyophilized. For in vitro assays, the microorganism was heat killed.

Cell lines

Mouse macrophage RAW264.7 cells were obtained from the Korean Cell Line Bank. Cells were cultured at 37°C in DMEM (WelGENE, Daegu, Korea) containing 10% fetal bovine serum (Sigma, St Louis, MO, USA) and 1% antimycotic-antibiotic (Gibco-BRL, Grand Island, NY, USA) in a humidified atmosphere of 5% CO₂.

Animal experiments: Treatment protocol

Viable L. citreum was suspended in 9% skim milk to 2 × 10⁸CFU/ml. Each mouse received this suspension by oral gavage in a volume of 100 μl per animal three times per week. Untreated and control mice were given an equal volume of skim milk. Each experimental group consisted of seven to eight mice.

Mice were sensitized by i.p. injection with 1 μg OVA grade V (Sigma) dissolved in a mixture of an equal volume of Al(OH)₃ gel (alum) and PBS once per week for 3 weeks. Untreated mice were injected with PBS.

Experiments consisted of the pre-sensitization and post-sensitization protocols. For the effect of L. citreum on the induction of OVA-induced Th2 response (pre-sensitization), oral administration began 1 week before the first sensitization and lasted until the third sensitization. For its effect on the effector phase of OVA-induced Th2 response (post-sensitization), L. citreum treatment was applied immediately after the final sensitization and continued for 2 weeks. By the end of the post-sensitization experiment (on days 23, 25 and 28), mice received 20 μl of 0.01% of OVA solution to each nostril. Mice were killed at the end of the experiments. Serum and spleens were obtained from each mouse individually. The procedures are summarized in Figure 1.

In vitro OVA restimulation of in vivo sensitized splenocytes

Splenocytes from OVA-sensitized mice were prepared by disrupting the spleen between glass slides in RPMI-1640 (WelGENE) with 10% FBS and 1% antimycotic/antibiotics. After a 10-min centrifugation at 150 ×g to separate cells from debris, cells were washed in complete medium, followed by lysis of erythrocytes. Splenocytes were resuspended and adjusted to 5 × 10⁶ cells/ml and cultured for 72 hr with OVA (1 mg/ml) at 37°C in a 5% CO₂ incubator.

In vitro cell culture for cytokine analysis

Splenocytes from naïve Balb/c mice were isolated and stimulated with L. citreum. Cell supernatant and mRNA were obtained at 24 hr. Peritoneal macrophages were obtained from C57BL/6 mice 3 days after i.p. injection of 2 ml sterile thioglycollate medium (DIFCO). Cells were resuspended in complete DMEM and seeded at a density of 5 × 10⁵/ml. After 90 min, non-adherent cells were removed. To assess NF-κB or MAPK-dependent cytokine production, the adherent cells were incubated with various doses of pharmacological inhibitors for NF-κB (sulfasalazine) (Sigma), p38 (SB230580) (Promega, Madison, WI, USA), ERK1/2 (U0126) (Sigma) and JNK (SP600125) (Sigma) for 30 min before stimulation with the bacteria. Sulfasalazine was dissolved in NH₄OH solution and the rest of the inhibitors were dissolved in dimethylsulfoxide (DMSO). Control cells (0 μg/ml) were treated with an equal volume of DMSO or NH₄OH solution. To determine IκB degradation and phosphorylation of p38, ERK1/2 and JNK, RAW264.7 cells (3 × 10⁶ cells/ml) were stimulated with lactic acid bacteria for various time points.

ELISA

The levels of IL-4, IL-5, IFN-γ, IL-12, IgE and IgG2a were measured using a commercial OptEIA ELISA set (Pharmingen, San Diego, CA, USA). IgG1 was measured using capture antimouse IgG1 (A85-3) and biotinylated detection anti-mouse IgG1 (A85-1) (BD Pharmingen). Purified mouse IgG1 isotype (BD Pharmingen) was used as standard. Each cytokine and antibody concentration was calculated from the standard curve.

Real-time RT-PCR

Total RNA was isolated with an RNeasy Mini Kit (QIAGEN, Hilden, Germany) and cDNA was reverse-transcribed by a Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Oligonucleotide primers were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA). The GAPDH gene was used as an endogenous control to normalize the expression of target genes. Quantitative real-time PCR was carried out in triplicate using a 96-well optic tray on an ABI PRISM 7300 sequence detector (Applied Biosystems). Negative controls lacking template RNA were included in each experiment. The expressions of each mRNA were quantified by relating the PCR threshold cycle obtained from the samples to amplicon-specific standard curves and normalized to each GAPDH gene.

Western blot analysis

Cells were harvested, washed twice with ice-cold PBS and lysed on ice for 20 min using PRO-PREP protein extraction solution (iNtRon, Seongnam, Korea) containing phosphatase inhibitor (Sigma). Whole-cell lysates were centrifuged at 12 000 ×g at 4°C for 10 min and the supernatants obtained. Samples were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk in TBS-Tween (0.1%) for 1 hr and then incubated with polyclonal antibody for IκBα and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p38, JNK, ERK, phospho-p38, phospho-JNK and phospho-ERK (Cell Signaling, Beverly, MA, USA) overnight at 4°C. After washing three times with TBS-Tween, the membranes were incubated with secondary horseradish peroxidase-conjugated antirabbit antibody (Santa Cruz) for 1 hr. Band detection was conducted using an enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Uppsala, Sweden).

Statistical analysis

Comparisons between groups were determined by anova followed by Dunnet's post-hoc test. Calculations were carried out using SPSS version 12. P values less than 0.05 were considered significant.

Effect of L. citreum on the serum antibody response and splenocyte cytokine production from pre-sensitized mice

Allergic response consists of systemic response in the induction phase and local inflammation in the effector phase. First, we tried to measure the effect of L. citreum on the induction phase of OVA-induced Th2 response. Balb/c mice were orally given L. citreum prior to sensitization. This intervention lasted until the final sensitization and serum and splenocytes were obtained at the end of the experiment (Fig. 1) OVA-sensitized mice developed a typical Th2 response characterized by higher levels of serum IgE and IgG1. A significant reduction was observed in L. citreum-treated mice (Fig. 2a,b). Subsequently, in order to determine whether these inhibitions were indicative of overall B-cell suppression or the modulation of Th1/Th2 responses, we analyzed the levels of serum IgG2a, a Th1-type antibody. Surprisingly, a significant reduction in serum IgG2a was also elicited (Fig. 2c), suggesting that such inhibition did not happen selectively in IgE-producing B cells. We further investigated the production of Th1 and Th2 cytokines from OVA-restimulated splenocytes isolated from pre-sensitized mice. The immunomodulation of Th1/Th2 cytokine production was observed in the L. citreum-treated group: the secretion of IFN-γ was enhanced, concomitant with a decrease in IL-4 and IL-5 (Fig. 2d,e,f). These data indicate that the IgE inhibition by L. citreum does not appear to be due to a direct effect on the modulation of antigen-specific Th1 and Th2 responses.

Effect of L. citreum on the serum antibody response and splenocyte cytokine production from post-sensitized mice

Next, we wanted to see whether L. citreum could inhibit IgE production during the effector phase of the OVA-induced Th2 response. As shown in Figure 1, L. citreum treatment began right after the final sensitization until the end of the experiment for 2 weeks, which included OVA challenge. Like the effects observed with the pre-sensitization model, this intervention strongly inhibited the serum levels of IgE, IgG1 and IgG2a altogether, indicating that L. citreum treatment at post-sensitization also played an inhibitory role in B-cell responses (Fig. 3a,b,c). With regard to antigen-specific Th1/Th2 cytokine production, a higher level of IFN-γ was observed, but those of IL-4 and IL-5 were rather increased, although they did not reach statistical significance (Fig. 3d,e,f). Results from our two protocols suggest that regardless of the time points at which mice were sensitized, the mechanism behind the serum IgE downregulation by L. citreum might be associated with pan-B cell suppression, but not a direct result of the modulation of Th1/Th2 cytokine production.

Effect of L. citreum on the induction of Th1 cytokines in mouse splenocytes in vitro

In order to further characterize the ability of L. citreum to induce Th1 cytokines, mouse splenocytes were stimulated with the microorganism for 24 hr in vitro. We used heat-killed L. citreum in order to keep the cell count constant throughout all the in vitro assays. IL-12 and IFN-γ were examined at the protein and mRNA levels. IL-12 is a heterodimer of two chains of p40 and p35. Its biologically active form is p70 and expression of the p40 subunit reflects the production of IL-12 p70 while the mRNA of the p35 is relatively constant (20). Therefore, we measured IL-12 p70 and IL-12 p40 by ELISA and real-time RT-PCR, respectively. The peak of IL-12 expression occurs prior to that of IFN-γ (9). As expected, the production of IL-12 in response to L. citreum at 24 hr was more outstanding than that of IFN-γ, whereas at the mRNA level, the transcriptional activity of IFN-γ was stronger than that of IL-12 at the same time point (Fig. 4a,b). T-bet is involved in the inhibition of IgE in B cells as well as the upregulation of IFN-γ in T cells (21, 22). The induction of T-bet by L. citreum was also detected as shown by real-time RT PCR (Fig. 4b).

The expression of IFN-γ by lactic acid bacteria depends on the production of IL-12 by macrophages and dendritic cells, as the removal of these cells abrogates IFN-γ (8, 9). In an effort to identify whether L. citreum-induced IL-12 was necessary for IFN-γ production, we treated splenocytes with neutralizing anti-IL-12 mAb in the presence of L. citreum for 24 hr. Consistent with previous reports, the IFN-γ release by L. citreum was clearly decreased, but not completely (Fig. 4c). These results partly confirmed the IL-12 dependence of IFN-γ in response to the microbial stimulation.

Effect of L. citreum on NF-κB and MAPK signaling in IL-12 production in macrophages

We tried to determine the role of the NF-κB and MAPK pathways in L. citreum-induced IL-12 production in macrophages. Peritoneal macrophages from C57BL/6 mice were obtained and cultured with the bacteria in the presence of varying concentrations of specific pharmacological inhibitors for NF-κB (sulfasalazine), ERK1/2 (U0126), p38 (SB203580) and JNK (SP600125). Our results showed that these inhibitors inhibited IL-12 production in a dose-dependent manner, indicating that NF-κB, p38 and JNK clearly participated in the cytokine production (Fig. 5a). ERK1/2 seemed to play a marginal role, as the inhibition was only observed at the highest dose tested (50 μM). Next, western blotting was carried out in order to identify whether L. citreum was able to directly activate these proteins. Mouse macrophage RAW264.7 cells were stimulated with the bacteria for various time points. IκB degradation, which is indicative of nuclear translocation of NF-κB by L. citreum, was detected (Fig. 5b). We also found that p38, JNK and ERK1/2 were activated. From these results, it can be concluded that activation of p38, JNK and NF-κB signaling pathways are mainly involved in IL-12 production in L. citreum-treated macrophages.