Synergistic oligodeoxynucleotide strongly promotes CpG-induced interleukin-6 production
Bacterial genomes span a significant portion of diversity, reflecting their adaptation strategies; these strategies include nucleotide usage biases that affect chromosome configuration. Here, we explore an immuno-synergistic oligodeoxynucleotide (iSN-ODN, named iSN34), derived from Lactobacillus rhamnosus GG (LGG) genomic sequences, that exhibits a synergistic effect on immune response to CpG-induced immune activation.
The sequence of iSN34 was designed based on the genomic sequences of LGG. Pathogen-free mice were purchased from Japan SLC and maintained under temperature- and light-controlled conditions. We tested the effects of iSN34 exposure in vitro and in vivo by assessing effects on mRNA expression, protein levels, and cell type in murine splenocytes.
We demonstrate that iSN34 has a significant stimulatory effect when administered in combination with CpG ODN, yielding enhanced interleukin (IL)-6 expression and production. IL-6 is a pleotropic cytokine that has been shown to prevent epithelial apoptosis during prolonged inflammation.
Our results are the first report of a bacterial-DNA-derived ODN that exhibits immune synergistic activity. The potent over-expression of IL-6 in response to treatment with the combination of CpG ODN and iSN34 suggests a new approach to immune therapy. This finding may lead to novel clinical strategies for the prevention or treatment of dysfunctions of the innate and adaptive immune systems.
KeywordsLGG CpG-ODN iSN-ODN IL-6 Synergistic effect
- CpG ODN
- iSN ODN
Systematic-onset juvenile chronic arthritis
Lactobacillus rhamnosus GG
Pathogen-associated molecular pattern
Systematic-onset juvenile idiopathic arthritis
Tumor necrosis factor
Nucleic acids have been shown to be particularly potent molecular triggers of the innate immune response, not only providing a quick response against pathogens but also playing a role in shaping the adaptive immune response. These nucleic acids include CpG oligodeoxynucleotides (CpG ODNs) that are responsible for the immune stimulatory effect of bacterial DNA . Several studies have demonstrated that bacterial DNA, as well as synthetic CpG ODNs (ODNs containing unmethylated CG dinucleotides and a phosphorothioate or chimeric backbone that renders these molecules nuclease resistant), have potent immunostimulatory effects . In the present study, we investigated the immune response using ODN candidates from the Lactobacillus rhamnosus GG (LGG) genome. Notably, the genomes of diverse bacteria share DNA motifs that are rarely found in higher vertebrates [3, 4, 5]. These motifs include non-methylated CG dinucleotides that trigger cells expressing Toll-like receptor 9 (TLR9); this trigger results in the activation of natural killer (NK cells), B cells, monocytes, macrophages, and dendritic cells [2, 6, 7, 8, 9], yielding an innate immune response characterized by the production of Th-1 cells and proinflammatory cytokines [10, 11, 12].
It is speculated that some intestinal bacteria exert beneficial effects, whereas others demonstrate deleterious effects. LGG chromosomal DNA has been shown to be a potent inducer of splenic B cell proliferation, CD86/CD69 expression, and cytokine production in mouse  and an efficient suppressor of allergic activity . In addition, various bacterial strains (including Lactobacillus, Lactococcus, Bifidobacterium, Enterobacter cloacae, Bacteroides fragilis, Enterococcus faecalis, and Escherichia coli) have been shown to induce pro inflammatory mediators such as tumor necrosis factor (TNF) -α, interleukin (IL) -6, IL-12p70, and IL-23; these mediators may in turn be responsible for the induction and maintenance of chronic inflammatory responses [15, 16, 17, 18]. One component shared among these inducing organisms is the nature of bacterial DNA. Bacterial DNA constitutes a pathogen-associated molecular pattern (PAMP) that is recognized by the vertebrate immune system, leading in turn to coordinated immune responses comprising both innate and acquired immunity . Unmethylated CpG dinucleotides, which are present at high frequency in prokaryotic DNA but are rare in eukaryotic DNA [20, 21], are considered one such PAMP. Other research has suggested that bacterial DNA, alone or in combination with other bacterial products, triggers the release of IL-6, IL-12, interferon (IFN) -γ, and immunoglobulin (Ig) M . In this context, the ability of bacterial DNA to induce IL-6 is of special interest. In fact, overproduction of IL-6 in vivo has been shown to cause various clinical symptoms and abnormalities (e.g., spiking fever, skin rash, arthritis, pericarditis, hepatosplenomegaly, and growth retardation) in laboratory models, which may explain the manifestations observed in patients with various inflammatory diseases, including rheumatoid arthritis (RA) and systematic-onset juvenile idiopathic arthritis (soJIA) [23, 24]. Here, we show that stimulation of mouse splenocytes with the combination of CpG-ODN and an immuno-synergistic ODN (iSN-ODN, which we have designated iSN34) derived from LGG genomic sequences yields significant up-regulation of IL-6 expression. We further demonstrate that this combination treatment leads to increased IL-6 expression in vivo, and that this increased expression enhances clinical symptoms and abnormalities. Indeed, CpG-motif-containing ODNs are widely studied as promising adjuvants for vaccines against a range of diseases, including infections, cancer (including kidney, skin, breast, uterine and immune malignancies [25, 26]), and allergies. Thus, our results are consistent with those previous studies; our observation of selective enhancement of IL-6 expression suggests that synergistic ODNs might be useful as vaccine adjuvants.
Pathogen-free female C57BL/6 mice (4 weeks of age) were purchased from Japan SLC (Shizuoka, Japan) and maintained under temperature- and light-controlled conditions. Mice were provided with ad libitum access to a standard diet of Labo MR Breeder (Nihon Nosan Co., Kanagawa, Japan) and sterile water. Mice were 6 to 7 weeks of age at the start of the study.
Cells and cell culture
Splenocytes were prepared using standard methods [32, 33]. and were then cultured in triplicate or quadruplicate wells of a 24-well plate (Nalge Nunc International K.K., Tokyo, Japan) at 2 × 106 cells/well in volumes of 1 mL/well of RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (Sigma-Aldrich), 100 U/mL penicillin, 100 μg/mL streptomycin, 25 mM HEPES, 1.0 mM sodium pyruvate, nonessential amino acids, and 0.0035% 2-mercaptoethanol (2-ME).
For analysis of gene expression, total RNA was isolated from ODN-stimulated mouse splenocytes and treated with DNase I (Macherey-Nagel GmbH & Co., Duren, Germany) for 15 min at room temperature; the nuclease was then heat-inactivated by incubation of the mixtures at 70 °C for 15 min [29, 34, 35, 36]. Thereafter, cDNA was prepared by reverse transcription from 1 μg of total RNA per sample using the prime Script® RT reagent kit (TaKaRa Bio, Inc., Tokyo, Japan). Equal volumes of cDNA were used for quantification of various cytokine cDNAs via real-time quantitative PCR (qPCR) using a Thermal Cycler Dice® Real Time System (TaKaRa Bio, Inc., Tokyo, Japan). The qPCR analyses were performed with SYBR Premix Ex Taq (TaKaRa Bio) using gene-specific primers, as described previously . Primers for the β-actin- and IL-6-encoding genes were purchased from TaKaRa Bio. As a control, poly (A)+ RNA samples were used as templates to check for the presence of contaminating genomic DNA. Each pair of gene-specific primers included one primer designed to span an exon-intron junction, and the other designed to span the actual exon-intron boundary. The sensitivity of the reaction and amplification of contaminating products, such as self-annealed primers, were evaluated by amplifying serial dilutions of cDNA. For cross-sample comparison of results obtained following various treatments, levels of cytokine-encoding mRNA were first normalized to those of β-actin-encoding mRNA. Data are shown as the mean + standard deviation (SD) of one experiment representative of three independent experiments that yielded similar results.
Splenocytes were prepared using standard methods . Cells were cultured in a 24-well plate (Nalge Nunc International K.K., Tokyo, Japan) at 1 × 107 cells/well in volumes of 1 mL/well of complete RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 25 mM HEPES, 1.0 mM sodium pyruvate, nonessential amino acids, and 0.0035% 2-ME. Cells were incubated in the presence of 3 μM iSN34 for 24 h at 37 °C in a 5% CO2 environment. Cells were then washed with fresh medium to remove the iSN34. Cells were resuspended in fresh medium supplemented with 3 μM CpG-B (ODN 1555) for 6 h and cytokine expression was detected by qPCR as described above. Data are shown as the mean + SD of one experiment representative of three independent experiments that yielded similar results.
IL-6 levels in cell culture supernatants after 48 h of various treatments were quantified using a commercially available ELISA kit (eBioscience Inc., San Diego, CA, USA) according to the manufacturer’s instructions.
Splenocytes (2 × 106 cells/well) were pre-incubated for 3 h in medium supplemented with either 0.625 μM iSN34 or an equivalent volume of water. Cells then were washed with medium to remove the ODNs, resuspended in medium containing 3.0 μM CpG-B (ODN 1555), and incubated for 12 h. After stimulation, cells were cultured for 4 h at 37 °C in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, 100 μg/mL of streptomycin, 10 μg/mL of brefeldin A, 2 μg/mL of ionomycin, and 20 ng/mL of phorbol 12-myristate 13-acetate. For intracellular staining, cells were fixed in 4% PFA for 15 min at room temperature, washed, and permeabilized by incubation for 15 min on ice; cells were then further incubated with phycoerythrin-labeled anti-mouse IL-6 antibody (Biolegend). Splenocytes were first stained with anti-mouse IL-6 antibody for 60 min on ice. Cells were washed and the percentages of CD19+ IL-6+ cells were determined using FACS Calibur (BD Biosciences). Data were acquired and analyzed using Flow Jo software. All analyses were carried out at least in triplicate; representative results are presented.
In vivo study
In vivo experiments employed 4-week-old C57BL/6 female mice obtained and maintained as described above. After a preliminary acclimatization period of 2 weeks, mice (6 weeks of age) were sensitized by a total of 3 intraperitoneal (i.p.) injections (administered once every other week) of 200 μL of phosphate-buffered saline (PBS) + PBS, PBS (100 μL) + CpG-B (100 μg), iSN34 (20 μg) + PBS, or iSN34 (20 μg) + CpG-B (100 μg). For this study, MsST was used as the CpG-B. MsST is a strong immunostimulatory CpG ODN that is derived from the lacZ gene of Streptococcus thermophilus ATCC19258 and has an ability similar to that of the murine prototype CpG ODN (ODN 1555) to induce inflammatory cytokine production and cell proliferation . Mice were subjected to euthanasia and necropsy at 1 week after the final i.p injection (i.e., at 11 weeks of age). At sacrifice, we measured body weight, spleen weight, and spleen length, and collected blood for further analysis.
All statistical analyses were performed using a statistical software package (Prism 7, GraphPad, Inc., La Jolla, CA, USA). Two-tailed One-way ANOVA with a post-hoc Tukey-Kramer test was used to determine the significance of the differences in all experiments except for the body weight trends. Body weight gain changes were analyzed by Two-way ANOVA with post-hoc Bonferroni to compare treatment and time effects. Differences were considered significant at p < 0.05. Values for the in vivo experiment (spleen weight and spleen length) are presented using box and whisker plots. Other in vitro values are presented as means + SDs of three independent experiments (n = 9).
Immune synergistic activity of LGG ODN and expression of IL-6 mRNA by murine immune cells
Induction of IL-6 production by exposure to low-concentration iSN34
Enhancement of the IL-6+ subpopulation in murine splenocytes
Effect of i.p. injection of iSN34 + CpG
Our study revealed that iSN34 exhibits immune synergistic effects when combined with CpG-ODN. The activity of iSN34 was associated with multiple types of CpG-ODNs. We showed the synergistic activity of iSN34 + CpG at the level of IL-6 mRNA accumulation, IL-6 protein secretion, and IL-6+ cell production following in vitro exposure of cultured splenocytes. Among the tested ODNs, only iSN34 exhibited these effects, which were observed only when combined with CpG ODN. Previous work has shown that CpG ODN is an extremely potent stimulator of dendritic cells and macrophages, causing the induction of Th-1-associated cytokines such as IL-6 and IL-12 . In the present work, the ODN iSN34 was able (in combination with CpG) to stimulate the production of IL-6, suggesting that iSN34 would be a highly effective adjuvant for Th-1-mediated vaccines.
Thus, our results indicated that the combination of iSN34 + CpG strongly induces the expression of IL-6, and therefore might be useful for the prevention or treatment of diseases associated with inflammatory disorders, including RA, inflammatory bowel disease (IBD), multiple sclerosis, systematic-onset juvenile chronic arthritis (JCA), osteoporosis, and psoriasis [38, 39, 40, 41]. However, other work has shown that IL-6 acts as a potent stimulator of B-cell proliferation , plasma cell survival , and antibody production , effects that might be at odds with the prevention or treatment of some diseases. Further work will be needed to address the combination of these distinct effects.
We observed that iSN34 exhibited greater synergy when combined with CpG-B (Fig. 1b). On the other hand, iSN34 did not display synergy in combination with CpG-C. These results indicated that CpG-B is the appropriate synergistic inducer for use with iSN34. Our studies also showed that co-administration of iSN34 + CpG ODN had synergistic effects in a wash-out assay. Specifically, we demonstrated that splenocytes pretreated with iSN34 maintain the ability to stimulate the immune response even after the iSN34 is washed out, suggesting that the immune synergistic effect of iSN34 may be mediated via stimulation of cell signaling. Assessment of the immune synergistic effect of iSN34 + CpG-B by measuring IL-6 protein secretion (via ELISA) demonstrated that 0.63 μM iSN34 yielded peak synergistic efficacy under our experimental conditions.
CD19 is one of the most reliable biomarkers for B cells that can receive positive stimuli from a variety of cytokines, including IFN, IL-4 and IL-6; such stimulation leads to proliferation, differentiation, cytokine production, and other effector functions . In the present work, B cells exposed to the combination of CpG and iSN34 showed significant up-regulation of CD19 expression compared to controls. To clarify whether specific subpopulations of cells were selectively activated by iSN34, we further characterized IL-6producing splenocytes using flow cytometry. This flow cytometric analysis revealed that the proportion of IL-6secreting CD19+ B-cells in the spleen increased over two-fold in mice treated with iSN34 + CpG-B, compared to the levels seen in the control group (Fig. 3c). These findings could be relevant for the study of inflammation.
We also found that co-administration of iSN34 + CpG-B in vivo in healthy mice was sufficient to cause expansion of this cell type, yielding increased spleen weight and length. Notably, this treatment did not result in significant changes in terminal body weight compared to control groups, suggesting that iSN34 was not associated with gross toxicity (as might be implied by weight loss or attenuation of weight gain). We did, however, detect hypertrophy of the spleen in mice sensitized by i.p. injection with iSN34 + CpG-B. In previous work, we demonstrated that CpG-B is involved in activating the innate immune response and induces splenomegaly [29, 46]. Indeed, splenomegaly has been considered an important complication of acute and chronic disease for more than 100 years [47, 48, 49].
Notably, iSN34 does not provide significant synergistic induction of inflammatory cytokines (TNF-α, IFN-γ) other than IL-6 (data not shown). Given this observation, combined therapies incorporating iSN34 may significantly complement and further enhance the positive effects of mucosal immunity. In summary, our study demonstrated that iSN34 acts synergistically in combination with CpG ODN in the induction of IL-6. This multifunctional cytokine was originally identified as a T-cell-derived factor, and has subsequently been shown to induce the differentiation of activated B-cells into antibody-producing cells with biological activities that include the regulation of immune response, inflammation, and hematopoiesis . This synergy may represent a new modality for the treatment of systematic inflammatory disorders.
The new insights provided by this study suggest a new concept of synergistic ODN activity distinct from that described in previous ODN studies. However, the implications of this synergy for clinical symptoms or disease risk are unknown. These facts suggest that this immune synergy represents a wide-open research field with good scientific prospects, and could yield improvements in the prevention and treatment of immune disorders. However, further preclinical and clinical investigations will be needed to understand how specific targeting of the IL-6 pathway can be applied in disease treatment.
We thank Ms. Mizuki Yamada and Mr. Ryu Sugimura (Faculty of Agriculture, Shinshu University) for excellent animal care and technical support.
This work was supported by A-STEP (Adaptable and Seamless Technology Transfer Program through Target-driven R&D).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Design of the research, TSh; performing experiments, SN, YY, TOa; data interpretation, SN, TS; supplying materials, TSa; writing manuscript, SN, TSa. All authors read and approved the final manuscript.
All experimental procedures were carried out in accordance with the Regulations for Animal Experimentation of Shinshu University, and the animal protocol was approved by the Committee for Animal Experiments of Shinshu University. Based on national regulations and guidelines and in accordance with Law No. 105 and Notification No. 6, all experimental procedures were reviewed by the Committee for Animal Experiments and finally approved as No. 230030 by the president of Shinshu University.
Consent for publication
The authors declare that there are no conflicts of interest in this study.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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