A decoy oligodeoxynucleotide favors the differentiation of CpG ODN-induced B cells into IL-10-producing Breg-like cells over plasma cells by restoring IRF4/IRF8 imbalance

Abstract

The imbalanced expression of interferon regulatory factor (IRF) 4 and IRF8 in activated B cells significantly influences their differentiation and promotes the development of immune-related diseases. Restoring abnormal B cells to appropriate responses may treat these diseases. In this study, an oligodeoxynucleotide (ODN) S2, designed according to the consensus sequence recognized by IRFs in interferon-stimulated response elements, was used as an immunomodulator to investigate its effects on mouse splenic B cells stimulated with the TLR9 agonist CpG ODN, either alone or in combination with antigen, and to explore its underlying mechanisms. The results showed that S2 had a significant negative regulatory effect on CpG ODN induced B cell activation. It also significantly downregulated the production of IL-6 and the percentage of IL-6⁺ B cells in splenocytes stimulated by CpG ODN, but significantly upregulated the percentage of IL-10⁺ B cells. Interestingly, S2 impaired antibody production both in vitro and in vivo, but rescued mice from lethal inflammatory responses. Further studies showed that S2 could bind IRF4 and IRF8 with high affinity, slightly upregulate phosphorylated IRF4, reduce the expression and nuclear translocation of IRF8, and alter the proportion of IRF4⁺, IRF8⁺ or double-positive B cells in spleen cells induced by CpG ODN. These results suggest that S2 acts as a decoy directing some B cells to differentiate into IL-10-producing Breg-like cells rather than plasma cells by restoring the TLR9 signal-induced IRF4 and IRF8 ratio imbalance. This indicates its potential as an immunomodulator for the treatment of diseases associated with B-cell abnormalities.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-025-06033-9.

Introduction

B cells are traditionally known for their ability to secrete antibodies in the context of mediating adaptive immune responses. However, as a vital subgroup of lymphocytes, B cells, in addition to producing antibodies, play a pivotal role in immunomodulation by presenting antigens, secreting cytokines, and regulating the functions of other immune cells, acting as a bridge between adaptive and innate immune responses. B cells are a population comprising different subsets, which are influenced by their environment and the stimuli they are exposed to. B cells primarily originate in the bone marrow and mature in the periphery. Upon encountering antigens, they can be activated and differentiated into antibody-secreting cells such as plasma cells (PCs) and memory B cells. Additionally, a subset of B cells can differentiate into regulatory B cells (Bregs) in response to excessive inflammatory signals. Bregs are terms for all immune suppressive B cells that help maintain immune tolerance and limit excessive immune responses, thereby re-establishing immune homeostasis [1]. A functional hallmark of Bregs is the production of IL-10, and IL-10-producing B cells are often considered potential Breg-like cells. Furthermore, the activation and differentiation of B cells can be mediated not only by specific antigen signaling through the B-cell receptor (BCR), but also by non-specific signaling via pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs). Toll-like receptor 9 (TLR9), a PRR that is constitutively expressed in B cells, can be activated by agonists such as CpG oligodeoxynucleotide (CpG ODN). This activation promotes the proliferation and differentiation of B cells into antibody-secreting cells, up-regulates molecules involved in immune cell interactions, and enhances the secretion of pro-inflammatory cytokines (e.g., IL-6, TNF) or anti-inflammatory cytokines such as IL-10 [2, 3]. The activation and differentiation of B cells involve several members of the interferon regulatory factor (IRF) family, in particular IRF4 and IRF8. Therefore, changes in IRF4 and IRF8 signaling dynamically influence B cell activation and differentiation.

IRF4 and IRF8 are the two most structurally related members of the IRF family, which comprises nine members in mammals, IRF1-9, that were initially identified for their role in inducing the transcription of type I interferons-encoding genes [4]. All IRF proteins contain a conserved amino-terminal DNA-binding domain (DBD) that typically recognizes a DNA sequence element named the interferon-stimulated response element (ISRE) containing the IRF consensus sequence [(GAAA(G/C) (T/C) GAAA] [5, 6]. In addition, IRF4 and IRF8 have evolved distinct DNA-binding modes that enable them to co-assemble with transcription factors such as PU.1, Spi-B or BATF, through their IRF association domain (IAD) on composite DNA elements such as EICE or AICE [7]. Functionally, IRF4 and IRF8 are primarily involved in B cell activation and differentiation [8] and implicated in immunoregulation mediated by TLRs and other PRRs [9]. IRF4 is an unusual component of the gene regulatory network in activated B cells, where it acts as an obligate controller of opposing cellular states in a concentration-dependent manner. A high concentration of IRF4 drives the rapid generation of PCs [10], whereas a low concentration facilitates immunoglobulin class switch recombination [10, 11]. IRF4^−/− mice exhibit severely impaired B-cell activation and differentiation, accompanied by a profound reduction in serum antibodies of all isotypes [8]. Furthermore, heterozygous germline mutations in the DNA-binding domain of IRF4 cause combined immunodeficiency in humans [6, 12]. The expression of IRF4 is strongly upregulated upon co-stimulation of follicular B cells with CD40 [13]. Meanwhile, CpG ODN, a TLR9 agonist, upregulates CD40 expression on these cells and has also been shown to transiently enhance IRF4 expression while maintaining follicular B cell identity for up to 2 days. Co-stimulation with both CpG ODN and IFN-α further enhances IRF4 expression and promotes B cell differentiation into PCs by day 3 [14]. IRF8 can interact antagonistically with IRF4 through their shared sites in the cis-regulatory region during B cell activation [15]. Early on, it was found that IRF8 is constitutively expressed throughout B cell differentiation except in mature PCs [16]. During the pre-B to B cell transition, IRF8 function depends on its heterodimerization with IRF4 [17]. Among mouse and human B lineage cells, IRF8 is expressed at the highest levels in germinal center (GC) B cells and promotes GC formation by controlling the expansion and maturation of marginal zone and follicular B cells [18–20]. IRF8^−/− mice exhibit increased numbers of splenic B cells [21]. It was recently found that mice with B cell-specific deletion of Irf8 have an abundance of Bregs [22]. The regulation of B-cell development and differentiation involves mutual antagonism between IRF4 and IRF8, which operate through a concentration-dependent double-negative feedback loop that regulates distinct gene-expression programs during the initial developmental bifurcation of activated B cells [15]. Altered expression or imbalanced ratios of IRF4 and IRF8 in peripheral blood B cells are associated with clinical severity and circulating PC frequency in patients with myasthenia gravis (MG). In two MG subgroups, IRF8 expression is negatively correlated with these clinical parameters [23]. The molecular studies of an aberrant NOTCH2-BCR signaling axis in B cells from patients with chronic graft-versus-host disease (cGVHD) have revealed imbalanced expression of IRF4 and IRF8 [24]. Thus, targeting IRF4 and IRF8 may provide a strategy to fine-tune B-cell differentiation and modulate the outcomes of the B-cell immune responses.

The dysregulation of B cell function and tolerance can lead to a wide array of diseases, including autoimmune [25] and lymphoproliferative [26] disorders, and even sepsis [27]. Indeed, it has been shown that patients with sepsis have alterations in the number and function of B cells [28]. Consequently, B-cell-related immunotherapies for sepsis often focus on replenishing the numbers and restoring the functions of B cells. Given that B cells can differentiate into specific subsets with selective function and localization characteristics, immunomodulators targeting B cell activation and differentiation may have the potential to treat B cell dysfunction-associated diseases. For example, B1a cells, an innate-like cell population in mice, are crucial for the regulation of inflammation through their release of natural IgM and IL-10 [29]. IL-10⁺ B cells are critical for immune homeostasis and restrain excessive immune responses in settings such as infection, cancer, and inflammation [30]. For example, IL-10-producing B cells can protect B cell-specific TLR9-deficient NOD mice from type 1 diabetes (T1D) [31]. In this study, we found that S2 ODN, a self-designed non-CpG ODN, promotes the differentiation of activated B cells into IL-10-producing B cells rather than PCs. We propose that it may act as a decoy ODN that restores the imbalance in IRF4 and IRF8 ratios in TLR9 activated B cells. These findings suggest that S2 ODN has potential as an immunomodulator for the treatment of disorders associated with B-cell dysfunction, such as autoimmune and autoinflammatory diseases or excessive inflammatory response syndrome.

Materials and methods

Oligodeoxynucleotides and other reagents

The ODNs used in this study included CpG ODN 5805 (CpG5805 for short, a B-type CpG ODN) [32], ODN MS19 with GAAA repeats (an IRF5 decoy ODN) [33–35], ODN S2 with three GAAA motifs (5’-xxxGAAAxxxxGAAAxxxxGAAAx-3’), 5’-Biotin-labeled S2 (Biotin-S2), 5’-Biotin-labeled MS19 (Biotin-MS19) and 3’-Cy3- labeled S2 (S2-Cy3). All ODNs were synthesized by TaKaRa Biotechnology Company (Dalian, China) with nuclease-resistant phosphorothioate modification and diluted in sterile phosphate-buffered saline (PBS) prior to use.

Anti-mouse Fluorescence-labeled antibodies: CD19-PE (557399), CD19-FITC (553785), CD19-APC (550992), CD3e-APC (558257), CD11c-FITC (553801), NK1.1-APC (550627), TACI (CD267)-APC (562345), CD40-FITC (553790), IL-6-APC (561367), IL-10-PE (554467), and Mouse Th1/Th2/Th17 CBA Kit (560485) were from BD Biosciences (NJ, USA). TLR9-APC (FAB7960R) was from R&D Systems (MN, USA). IRF4-FITC (11–9858-82) and IRF8-APC (17–9852-82) were from Invitrogen (USA). Hepatitis B Vaccine (HBV) and Hepatitis B surface antigen (HBsAg) were from Aimen Hissen Vaccine Co., Ltd (Dalian, China). Skim milk and Urea were from Beijing Biotopped Science & Technology Co., Ltd (Beijing, China). Goat anti-Mouse IgG-HRP antibody (abs20001) was from Absin (Shanghai, China). Goat anti-Mouse IgM-HRP antibody (bs-0368G-HRP) was from Bioss (Beijing, China). OPD substrate was from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). RIPA buffer (P0013C), PMSF (ST507), Nuclear and Cytoplasmic Protein Extraction Kit (P0028), DAPI (C1005) and Brefeldin A (S1536) were from Beyotime (Shanghai, China). BCA protein assay kit (CW0014S) and Trizol reagent were from CW Biotech (Beijing, China). IRF4 Polyclonal antibody (11247-2-AP), Multi-rAb HRP-Goat anti-Rabbit Recombinant Secondary antibody (RGAR001) and Anti-GAPDH antibody (60004-1) were from Proteintech Group (USA). Anti-IRF5 (ab33478) antibody, and Anti-IRF8 (ab306552) antibody were from Abcam (UK). Phosphor-Tyrosine Monoclonal antibody (AB_2865096) was from ThermoFisher (MA, USA). Anti-PCNA antibody (HA601172) was from HUABIO (Hangzhou, China). Streptavidin Agarose Beads (S1638) were from Merck (Germany). Super ECL Detection Reagent was from Yeason Biotechnology (Shanghai, China). cDNA Synthesis Kit was from Transgen Biotech (Beijing, China). 4% paraformaldehyde was from Solarbio (Beijing, China). D-(+)-Galactosamine hydrochloride (D-GalN, G115553) was from Aladdin (Shanghai, China). Protein G Agarose Beads (37478) were from Cell Signaling Technology (USA). All primers used for RT-qPCR were synthesized and purified at Sangon Biotech Company (Shanghai, China) with HPLC-level purity of 99%. RPMI 1640 medium was from Gibco (Grand Island, NY, USA), Fetal bovine serum (FBS) was from Biological Industries, TBD (Tianjin, China).

Mice

Eight-week-old female Balb/c mice were purchased from Yisi Laboratory Animal Technology Co., Ltd. (Changchun, China). The mice were housed in microisolator cages under laminar airflow conditions and had free access to food and water throughout the study. All animal experimental procedures, including spleen isolation, were approved by the Animal Ethics Committee of the College of Basic Medical Sciences, Jilin University (Approval No. 2024 − 295). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80 − 23, revised 1996) and the institutional guidelines of Jilin University. This study was also approved by the Scientific Investigation Board of Science and Technology of Jilin Province, China.

For vaccination, mice were intramuscularly injected on day 0 and day 28 with 1 µg of hepatitis B vaccine (HBV) either alone or accompanied by CpG5805 (5 µg), or by CpG5805 (5 µg) in combination with MS19 or S2 (25 µg each).

To establish and treat the mouse model of systemic inflammatory response syndrome (SIRS), mice were first sensitized by intraperitoneal (i.p.) injection of 16 µg D-GalN and, after 1.5 h, randomly assigned to experimental groups. SIRS was then induced via i.p. injection of CpG5805 at doses of 5, 10, or 20 µg per mouse. Based on preliminary screening, a dose of 10 µg CpG5805 per mouse was selected for subsequent experiments, as it effectively triggered SIRS without causing premature mortality or spontaneous remission. For treatment, D-GalN-sensitized mice received CpG5805 (10 µg/mouse) combined with either S2 or MS19 (50 µg/mouse), administered intraperitoneally. Mouse survival was monitored and recorded.

Cells and cell culture

Naïve murine splenocytes were prepared by mechanically grinding isolated mouse spleens and filtering the resulting suspension through a mesh screen. The cells were centrifuged at 600×g for 5 min, and the supernatant was discarded. The pellet was then resuspended in ice-cold ACK buffer for 2 min to lyse erythrocytes, after which sterile PBS containing 2% fetal bovine serum (FBS) was added to terminate the lysis reaction. Following another centrifugation step at 600×g for 5 min, the splenocytes were resuspended for subsequent experiments.

To evaluate the response of splenic B cells to various stimuli in vitro, the isolated splenocytes were cultured at a density of 1 × 10⁶ cells/ml in complete RPMI 1640 medium, supplemented with 10% (vol./vol.) heat-inactivated FBS and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Cultures were maintained at 37 ℃ in a humidified incubator with 5% CO₂.

The murine B cell line A20 was obtained from Haixing Biosciences (Suzhou, China). Cells were cultured in RPMI 1640 medium supplemented with 10% (vol./vol.) heat-inactivated FBS and antibiotics at 37 ℃ under a humidified atmosphere of 5% CO₂. These cells were subsequently used for pull-down assays.

Cell experiments

Mouse splenocytes were stimulated with varying doses of CpG5805 for different durations to induce splenic B cells activation. Based on initial screening, 0.5 µg/ml CpG5805 was selected for a 24 h stimulation to establish the experimental platform for B cells activation. Using this platform, the optimal dose of S2 required to inhibit B cells activation was determined to be 8 µg/ml. To evaluate the influence of BCR signaling on the TLR9-mediated response, HBV containing 0.4 µg/ml HBsAg was added to the splenocyte cultures. For the S2 tracing experiments, S2-Cy3 (1 µg/ml) was applied to the cells for the indicated durations. After incubation, cells were harvested and subjected to the following analyses: flow cytometry for assessing cell activation and S2-Cy3 uptake; immunofluorescence imaging for examining cellular morphology and S2-Cy3 localization; cytometric bead array (CBA) for quantifying cytokine secretion; dot blotting for detecting immunoglobulin production; western blotting for measuring specific protein expression levels; and RT-qPCR for evaluating the mRNA expression of genes of interest.

Flow cytometry

Cells were stained with fluorochrome-conjugated antibodies diluted in PBS according to the manufacturer’s instructions. Each sample consisted of approximately 10⁶ cells. For surface antigen staining, cells were incubated for 30 min on ice in the dark with the following fluorescently labeled antibodies: CD19-PE/FITC/APC, CD3e-APC, CD11c-FITC, NK1.1-APC, TACI-APC, CD40-FITC, or TLR9-APC. For intracellular staining, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, and subsequently stained in the dark for 30 min with specific antibodies against IRF4-FITC, IRF8-APC, IL-6-APC, or IL-10-PE. For cytokine detection (IL-6 and IL-10), Brefeldin A was added to the culture medium 4 h prior to cell harvesting to inhibit protein secretion and retain cytokines intracellularly. Following staining, all samples were washed with PBS and analyzed using an Accuri C6 flow cytometer (BD Biosciences, NJ, USA).

Immunofluorescence detection

For morphological analysis, cultured splenocytes were centrifuged onto glass slides, fixed with 4% paraformaldehyde for 15 min at 4 ℃, and stained with a PE-conjugated CD19 antibody for 1 h at 37 ℃ in the dark. Samples were then visualized using a BX53 fluorescence microscope (Olympus, Tokyo, Japan).

For the S2-Cy3 tracing assay, cells were first incubated with a fluorescently labeled CD19-FITC antibody at 4 ℃ for 30 min to label the cell surface. The cells were then centrifuged onto glass slides, fixed with 4% paraformaldehyde for 15 min at 4 ℃, and subsequently incubated with 1 µM DAPI for 5 min at room temperature (RT). Finally, the slides were cover-slipped and imaged using a confocal microscope (Olympus, Tokyo, Japan). Image analysis was performed with FV10-ASW 4.2 Viewer software (Olympus, Tokyo, Japan).

Reverse transcription & quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from cells using Trizol reagent, and then reverse-transcribed into cDNA using a cDNA synthesis kit. Quantitative Polymerase Chain Reaction (qPCR) was performed using a two-step SYBR Green assay (G31227; Transgene Biotech) on a Step One™ real-time PCR system (Applied Biosystems, Foster City, CA, USA), and the target genes were amplified by the specific primers as follows (forward and reverse): Actb, 5′- GATCAAGATCATTGCTCCTCCTG-3′ and 5′-AGGGTGTAAAACGCAGCTCA-3′, Nf-κb, 5′- GCATTCTGACCTTGCCTAT-3′ and 5′- CCAGTCTCCGAGTGAAGC-3′, Irf1, 5′-ATGCCAATCACTCGAATGCG-3′ and 5′-TTGTATCGGCCTGTGTGAATG-3′, Irf2, 5′-ACTGGGCGATCCATACAGGAA-3′ and 5′-GTAGACTCTGAAGGCGTTGTTT-3′, Irf3, 5′ -GAGAGCCGAACGAGGTTCAG-3′ and 5′-CTTCCAGGTTGACACGTCCG-3′, Irf4, 5′-CTTTGAGGAATTGGTCGAGAGG-3′ and 5′-GAGAGCCATAAGGTGCTGTCA-3′, Irf5, 5′-GGTCAACGGGGAAAAGAAACT-3′ and 5′-CATCCACCCCTTCAGTGTACT-3′, Irf6, 5′- CCAACAAGAGGAAGAAAACACC-3′ and 5′- GCCATCGTACATCAAGTTGAAC-3′, Irf7, 5′- TCCCAGACTGCCTGTGTAGA-3′ and 5′- ATCCAGATCCCTACGACCGA-3′, Irf8, 5′- CGGGGCTGATCTGGGAAAAT-3′ and 5′- CACAGCGTAACCTCGTCTTC-3′, Irf9, 5′-AAGCAAGACTTCCGAGAGGAC-3′ and 5′- TGGTTGGTTAGGGAGGGTTCC-3′, Prdm1, 5′- GCATCCTTACCAAGGAACCTG-3′ and 5′- TGGTGGAACTCCTCTCTGGAAT-3′, Il-6, 5′- GCCCACCAAGAACGATAGTCA-3′ and 5′- AGACAGGTCTGTTGGGAGTG-3′, Tnf-α, 5′-GACCCTCACACTCAGATCATCT-3′ and 5′-TCCCTTCACAGAGCAATGACTC-3′, Il-10, 5′-TGGACAACATACTGCTAACCGA-3′ and 5′- TCCTGAGGGTCTTCAGCTTCT-3′. The resulting cDNAs were amplified for one cycle at 95 ℃ (30 s) followed by 40 cycles at 95 ℃ (5 s) and 64 ℃ (31 s). The expression levels of target mRNAs were normalized to Actb mRNA levels and analyzed using the 2^−∆Ct method. Each assay plate included negative controls with no template.

Cytometric bead array (CBA)

Concentrations of cytokines (IL-2, IL-4, IL-6, IL-10, TNF, IL-17, and IFN-γ) in the supernatant of cultured splenocytes were quantified using a mouse Th1/Th2/Th17 CBA kit. Briefly, 50 µl of supernatant was combined with 50 µl of mixed capture beads and 50 µl of PE detection reagent. The mixture was incubated gently in the dark at RT for 2 h. After incubation, samples were washed and acquired on an Accuri C6 flow cytometer. Data analysis was performed using FCAP Array software.

Dot blotting

The cultured supernatants were applied onto nitrocellulose (NC) membranes (Schleicher & Schuell) and allowed to air-dry. After blocking with Tris-buffered saline containing 5% skim milk at RT for 1 h, the membranes were incubated with goat anti-mouse IgG-HRP or goat anti-mouse IgM-HRP antibody at RT for 1 h. Following washing, the membranes were developed using Super ECL Detection Reagent. Finally, the membranes were imaged with a WEALTEC imaging system, and the gray values of the spots of interest were quantified using ImageJ software.

ELISA

Hepatitis B virus surface antigen (HBsAg), diluted in PBS, was coated onto 96-well plates at 0.5 µg per well, and the plates were incubated at 4 ℃ overnight. After blocking with PBS containing 5% skim milk at 37 ℃ for 2 h, appropriately diluted test sera were added to the plates and incubated at 37 ℃ for 1 h. Following three washes with PBST (PBS with 0.05% Tween 20), 100 µl of a suitably diluted goat anti-mouse IgG-HRP antibody solution was added to each well and incubated at 37 ℃ for 1 h. After additional washing, 50 µl of OPD substrate was added to each well to initiate color development. The reaction was allowed to proceed at 37 ℃ for 10 min in the dark and was terminated by adding 50 µl of 1.25 M sulfuric acid per well. The optical density (OD) value at 492 nm was measured using an ELISA microplate reader (Bio-Tek Instruments, VT, USA).

Pull-down assay

Biotin-labeled S2 and MS19 ODNs were diluted to 100 µM in sterile PBS. A volume of 14 µl of each biotinylated ODN was incubated with streptavidin-agarose beads (50 µl) for 1 h at RT. Beads were washed and incubated with lysates of A20 cells overnight at 4 ℃ and then washed with PBS, then eluted with 50 µl elution (2% SDS, 0.4 M urea). Specifically bound IRF4, IRF5, and IRF8 were detected by western blotting.

Co-immunoprecipitation

Cell lysates were incubated with a phosphor-tyrosine monoclonal antibody at 4 ℃ overnight, after which protein G agarose beads were added and the mixture was incubated at 4 ℃ for an additional 2 h. After washing with PBS, bound proteins were eluted with 5×SDS loading buffer and subjected to western blotting analysis.

Western blotting

Total protein samples were extracted by lysing cells in cold RIPA buffer supplemented with 0.1 mM PMSF. Nuclear proteins were isolated from total protein extracts using a Nuclear and Cytoplasmic Protein Extraction Kit. Protein concentrations were determined with a BCA protein assay kit. Subsequently, proteins were separated by 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with Tris-buffered saline containing 5% skim milk for 1 h at RT, the membranes were incubated overnight at 4 ℃ with primary antibodies against IRF4, IRF5, IRF8, and GAPDH (for total protein) or PCNA (for nuclear protein). Following three washes with TBST (containing 0.05% Tween 20), the membranes were incubated for 1 h at RT with corresponding HRP-conjugated secondary antibodies, either goat anti-rabbit or goat anti-mouse IgG. The subsequent steps were consistent with those described for the dot blotting procedure.

Statistical analysis

Data are shown as mean ± SD. All calculations and statistical analysis were performed using GraphPad Prism 7.0 (GraphPad Software, USA) for Windows. Comparisons between groups were conducted using unpaired t-tests. A p < 0.05 was regarded as statistically significant. All experiments were performed at least three times.

Results

An oligodeoxynucleotide capable of inhibiting CpG ODN-induced B-cells activation

To find out whether oligodeoxynucleotides (ODNs) containing GAAA motif(s) could negatively regulate the immune response of B cells, we selected the self-designed ODN S2, and used CpG5805 to induce the activation of mouse splenic B cells in vitro as the research platform. By stimulating mouse splenocytes with different doses of CpG5805 for different times, we found that CpG5805 significantly upregulated the expression of CD40 on splenic B cells and the percentage of CD40⁺ B cells at each dose used (Fig. 1a). When CpG5805 (0.5 µg/ml) stimulated mouse splenocytes for 20 h or longer, it could up-regulate the percentage of CD40⁺ B cells to the maximum (Fig. 1b). Accordingly, we used 0.5 µg/ml CpG5805 to stimulate mouse spleen cells for 24 h as an in vitro research platform to detect the effect of S2 at different doses on the activation of splenic B cells induced by CpG5805. Compared with CpG5805 alone, there was no significant difference in the number and percentage of splenic B cells in the presence of CpG5805 combined with each dose of S2. However, some doses of S2, when combined with CpG5805, significantly downregulated the percentage of CD40⁺ B cells and CD40 expression levels. Since 8 µg/ml S2 reduced the percentage by 50%, it was chosen for further investigation (Fig. 1c). Subsequently, we examined the effect of S2 at 8 µg/ml on B cells and other cells in the splenocytes. After determining that the majority of cells in splenocytes were CD19⁺, CD3e⁺, CD11c⁺ and NK1.1⁺ cells in turn, we found that S2 mainly affected the proliferation and activation of CD19⁺ B cells induced by CpG5805, while no apparent difference was observed in the counts of CD3e⁺ and CD11c⁺ cells (Fig. 1d). We also demonstrated in vitro that S2 had no effect on the activation of CAL-1 cells (a human plasmacytoid dendritic cell line) and CD11c⁺ splenocytes induced by CpG5805 (Fig. S1). This suggests that S2 may be more inclined to regulate B cells activated by CpG ODN. The inhibitory effect of S2 on CpG5805-induced B cells activation was also confirmed by observation under fluorescence microscopy, in which S2 reduced the volume of B cells enlarged by CpG5805 (Fig. 1e). The above findings indicate that S2 downregulates CpG5805-induced CD40 expression on splenic B cells, while showing no significant impact on the population of CD19⁺, CD3e⁺, or CD11c⁺ splenocytes.

Fig. 1.

CpG5805 induced mouse splenic B-cells activation can be inhibited by S2 ODN with GAAA motifs in vitro. Mouse spleen cells were used to test the induction of CpG ODN 5805 (CpG5805) on splenic B-cells activation and S2 ODN on its inhibition for the CpG5805-induced B-cells activation. The splenocytes used in experiments were isolated from spleens of naïve mice and detected by flow cytometry or fluorescence microscope after the experiment. (a) Dose curve of CpG5805 stimulating the mouse splenic B-cells activation. (b) Time curve of CpG5805 stimulating the mouse splenic B-cells activation. (c) Dose curve of S2 inhibiting the activation of mouse splenic B cells induced by CpG ODN. (d) Effects of S2 on CpG5805-induced mouse splenic B cells and other cells. (e) Morphological images of B cells treated with CpG5805 alone or in combination with S2. Splenocytes were surface-stained with PE-conjugated anti-CD19 antibody and observed under a fluorescence microscope. Cells exhibiting orange fluorescence on the surface were identified as CD19⁺ B cells. These B cells were selected and imaged. A representative cell from each group of cell images was magnified, and its diameter was marked with a double-arrow dashed line. The scale bar in the enlarged image represents 5 μm. ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001; ^****, p < 0.0001

S2 ODN enters mouse spleen cells to interfere the mRNA transcription of IRFs during CpG ODN stimulation

It is already known that TLR9 signaling involves several transcription factors such as its downstream NF-κB and IRF family members, and Blimp-1 is a transcription factor directly involved in B cell response. To find out possible targets of S2 for its negative regulation on B cells responses to CpG ODN, mRNA levels of Nf-κb, Prdm-1 (Blimp-1 encoding gene), and Irf1-9 in mouse splenocytes stimulated by CpG5805 or CpG5805 combined with S2 were detected by RT-qPCR. The results showed that the mRNA levels of Nf-κb and Prdm-1 were up-regulated by CpG5805 at some time points, but the inhibitory effect of S2 to those was not obvious (Fig. 2a), suggesting that these two transcription factors may not be the main participants in the role of S2. When analyzing the mRNA levels of Irf1-9, we found that CpG5805 only significantly upregulated the mRNA levels of Irf4, Irf5 and Irf8. Under the induction of CpG5805, the mRNA level of Irf4 only increased in the early stage, the mRNA level of Irf5 showed a wavy increase and decrease, and the mRNA level of Irf8 was always increasing. S2 down-regulated CpG5805-induced Irf4/5/8 mRNA levels to different degrees at different time points (Fig. 2a). These findings implicate that IRF4, IRF5 and IRF8 may serve as the key players in S2-mediated suppression of CpG ODN-induced splenocyte responses.

Fig. 2.

Effect of S2 on the expression of Nf-kb, Prdm1 and Irf1-9 mRNA in CpG5805-induced splenocytes and its tracing of entering the cells. (a) The mRNA levels of Nf-kb, Prdm1 and Irf1-9 in splenocytes induced by CpG5805 alone or combined with S2. (b) The tracing of S2 entering splenocytes by flow cytometry. The splenocytes were surface stained with APC-labeled anti-CD19 mAb followed by detection on an Accuri C6 (BD Biosciences). c) The tracing of S2 entering into splenocytes by confocal immunofluorescence. The spleen cells were surface stained with FITC-labeled anti-CD19 mAb (green color) and nuclear stained with DAPI (blue color) after tracing cultured with S2-Cy3 (red color). After fixation, cell slides were prepared and examined under confocal microscope. The photos were taken at 600×. ^*, vs. Med; ^▽, vs. CpG; ^*/▽, p; 0.05; ^**/▽▽, p; 0.01; ^{***/▽▽▽}, p; 0.001; ^{****/▽▽▽▽}, p; 0.0001

Since IRF4, IRF5 and IRF8 are all transcription factors, S2 must be able to enter target cells containing these transcription factors if it is to act as a decoy. To determine whether S2 could enter target cells, we co-cultured mouse splenocytes with S2-Cy3 to trace S2 in vitro. Through dynamic detection by flow cytometry, we found that with the extension of culture time, the percentage of Cy3⁺ cells gradually increased in the splenocytes, of which Cy3⁺ B cells accounted for the largest proportion. After 6–20 h of the culture, the percentage of Cy3⁺ B cells in spleen cells reached 45–50%, while the percentage of Cy3⁺ non-B cells was only 10–30% (Fig. 2b), indicating that S2 can enter splenocytes in a non-specific way, mainly B cells. Then, we detected S2-Cy3 in splenic B cells cultured for 6 h and 20 h under immunofluorescence confocal microscopy. The results showed that S2-Cy3 (red color) was visible near the cell membrane and cytoplasm of CD19⁺ cells (green color) (Fig. 2c), which further confirmed that S2 could enter splenic B cells. The intracellular localization of S2 provides mechanistic support for its potential targeting of IRF molecules.

S2 ODN favors CpG ODN-induced B cells to differentiate into IL-10-producing Breg-like cells

Based on the above speculation and the knowledge that activated B cells can secrete cytokines, we examined the effect of S2 on the production of pro-inflammatory cytokine IL-6 and TNF, and anti-inflammatory cytokine IL-10 in mouse splenocytes or splenic B cells induced by CpG ODN. By dynamic detection ofIl-6,Tnf-αandIl-10mRNA levels in splenocytes by RT-qPCR, we found that S2 had a down-regulated effect on CpG5805-inducedIl-6mRNA levels at all time points. However, S2 showed down-regulation or up-regulation effects on CpG5805-induced Tnf-αandIl-10mRNA levels (Fig.3a). We then used Cytometric Bead Array (CBA) to dynamically detect IL-6, TNF and IL-10 levels in supernatants of cultured splenocytes at protein levels. It turned out that S2 still showed a significant down-regulation effect on CpG5805-induced IL-6 levels at all time points, but had little effect on the TNF level. Since the dynamic level of IL-10 in the supernatants was generally low, the effect of CpG5805 or S2 on it could not be determined (Fig.3b). These results suggest that S2 can negatively regulate the expression of pro-inflammatory factors in CpG ODN-induced spleen cells. To identify the population of proinflammatory and anti-inflammatory cytokine producing cells in spleen cells, we analyzed IL-6 and IL-10 levels in B cells and non-B cells in cultured splenocytes by flow cytometry. The results showed that CpG5805 mainly induced the expression of IL-6 in splenic B cells, the proportion of IL-6⁺CD19⁺ cells and the levels of IL-6 in CD19⁺cells were significantly increased during CpG5805 stimulation. CpG5805 also obviously upregulated the proportion of IL-10⁺CD19⁺cells, but not for the level of IL-10. S2 could significantly down-regulate the proportion of IL-6⁺CD19⁺cells and their IL-6 level up-regulated by CpG5805, but could obviously up-regulate the proportion of IL-10⁺ B/IL-10⁺non-B cells and their IL-10 levels (Fig. 3c). These results suggest that S2 can change the proinflammatory state of CpG ODN activated splenocytes, especially splenic B cells, into anti-inflammatory state. To further analyze the induction role of S2 on IL-10 expression in splenic B cells and non-B cells, we added a previously identified ODN (MS19) [33–36] that negatively regulates inflammatory response as control. The results showed that S2 caused approximately 10% of B cells and 5% of non-B cells in CpG5805-induced spleen cells to become IL-10-expressing cells, which was almost double the effect of CpG5805 alone, while MS19 did not have this effect (Fig. 3d). This indicates that S2 is more inclined to act on B cells and cause part of the B cells to differentiate into IL-10-producing Breg-like cells, while MS19 is not. We also confirmed this inference by detecting the expression of CD40, TACI and sTLR9 on splenic B cells. The results showed that S2 significantly decreased the percentage of CD40⁺/TACI⁺B cells induced by CpG5805 and the level of CD40/TACI on B cells, and obviously increased the percentage of sTLR9⁺B cells, while MS19 had no such effect (Fig. 3e).Using the same experimental platform as described above, we also observed that S2 affected the expression of CD40/TACI and sTLR9 on CpG5805-induced splenic B cells from C57BL/6 mice (Fig.S2), mirroring the effects observed in those of Balb/c mice. This finding suggests that the effect of S2 is not limited to Balb/c mice. According to the increase of sTLR9 and the decrease of CD40/TACI, both represent that B cells are in a state of inactivation or low response, even less likely to evolve into PCs, suggesting that S2 makes B cells enter a state of negative response to CpG ODN stimulation.

Fig. 3.

Effect of S2 on the expression of inflammatory cytokines in CpG5805-induced splenocytes. The mRNA level (a) and protein level (b) of IL-6, TNF-α and IL-10 in splenocytes and their supernatants. Mouse splenocytes were treated with CpG5805 alone or combined with S2 for various time and then used for detection of Il-6, Tnf-α and Il-10 at mRNA levels by RT-qPCR. The cultured supernatant of those splenocytes was used to detect IL-6, TNF and IL-10 at protein levels by CBA. (c) The expression of IL-6 and IL-10 in B cells and non-B cells of spleen cells. (d) Percentage of splenic B cells and non-B cells expressing IL-10. Note: MS19, an ODN with negative regulatory role of inflammatory response, was used as a control.(e)Percentage of CD40⁺/TACI⁺/sTLR9⁺ B cells and the levels of CD40/TACI/sTLR9 on B cells. Each point represents splenocytes from one mouse. ^*, vs. Med; ^σ, vs. CpG; ^*/▽, p < 0.05;^{**/ ▽▽}, p < 0.01; ^{***/▽▽▽},p < 0.001; ^{****/ ▽▽▽▽},p < 0.0001

S2 ODN is beneficial for alleviating lethal acute inflammatory response but not for antibody production

Based on the above results, S2 has a negative regulatory role on CpG ODN induced B cell activation which makes part of B cells prefer the differentiation of IL-10-producing Breg-like cells, it is speculated that S2 may have a suppressive effect on excessive inflammatory response. To test this hypothesis, we established a mouse model of systemic inflammatory response syndrome (SIRS) through intraperitoneal (i.p.) injection of D-GalN for 1.5 h followed by administration of 10 µg CpG5805 (Fig. 4a). We then observed the effects of S2 or MS19 on the survival of the model mice. The results showed that after the injection of CpG5805, the model mice began to die at 10 h, and died nearly 60% at 24 h. S2 could significantly prolong the lifetime and increase the survival rate of model mice. S2 delayed the onset of death in mice to 48 h after CpG5805 injection and maintained a mortality rate of 20% over the entire 72 h observation period. MS19 extended the onset of death in mice to 24 h after CpG5805 injection, but all mice died at this time point (Fig. 4b). This suggests that S2 can resist the excessive inflammatory response induced by CpG ODN in mice. This may be related to the effect of S2 to promote the differentiation of IL-10-producing Breg-like cells, as we found that S2 was resistant to CpG5805-induced lymphocytopenia and neutrophilia in peripheral blood of SIRS mice (Fig. S3).

Fig. 4.

Role of S2 in lethal acute systemic inflammatory response in mice and CpG5805-induced antibody production in vitro and in mice. (a) Survivals of mice with systemic inflammatory response syndrome (SIRS) induced by D-GalN combined with CpG5805. (b) Survival curves of SIRS-model mice treated with S2 or MS19. (c) Natural IgM and IgG levels in supernatants of spleen cells stimulated for 24 h by CpG5805 or CpG5805 + S2/MS19 were detected by dot blotting. (d) Natural IgM and IgG levels in supernatants of spleen cells stimulated for 24 h by HBV + CpG5805 or HBV + CpG5805 combined with S2/MS19 were detected by dot blotting. (e) The anti-HBsAg IgG levels in sera of mice immunized twice with HBV + CpG5805 or HBV + CpG5805 combined with S2/MS19 were detected dynamically by ELISA. Each dot represents the average antibody level in the serum of one mouse. Each diluted serum used for ELISA is duplicates. ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001; ^****, p < 0.0001

Since S2 tends to cause partial CpG5805-activated B cells to differentiate into IL-10-producing B cells, we wondered whether it also has a regulatory effect on CpG5805 alone or CpG5805 combined antigen-induced antibody production in vitro and in mice, as B cells may differentiate into antibody-producing cells, such as PCs, after activation. We firstly detected natural IgM and IgG in supernatants of splenocytes induced by CpG5805 alone or in combination with S2 or MS19 by dot blotting. We found that although CpG5805 did not increase IgM and IgG levels, S2 obviously reduced CpG5805-induced IgM and IgG levels, while MS19 did not (Fig. 4c). We then again used dot blotting to detect the effects of S2 or MS19 on natural IgM and IgG levels in supernatants of CpG5805 combined with hepatitis B vaccine (HBV)-induced splenocytes. The results showed that CpG5805 combined with HBV did not increase IgM levels, but significantly elevated IgG levels. S2 significantly reduced both IgM and IgG levels induced by CpG5805 combined with HBV, while MS19 only decreased IgM levels, and its intensity was much weaker than S2. S2 markedly suppresses CpG5805-induced IgM and IgG production, either alone or with antigens (e.g., HBsAg), while MS19 only weakly reduces IgM (Fig. 4d). This indicates that S2 modulates B cell differentiation or function via a mechanism different from MS19, inhibiting antibody-secreting cell formation and contributing to immune regulation. Since S2 can affect antibody production in vitro, does it have the same effect in vivo? Therefore, we used model mice immunized with HBV plus CpG5805 to observe the effect of S2 on anti-HBsAg specific IgG levels in sera of the model mice. In this experiment, we still used MS19 as the control. It was found that CpG5805 significantly increased the level of HBV-induced anti-HBsAg antibody in mice, while S2 down-regulated the levels of those antibodies but MS19 did not (Fig. 4e). This indicates that S2 has a negative regulatory role for TLR9 signaling or combined BCR signaling activated B cells to differentiate into antibody-producing cells, such as PCs.

S2 ODN caused the imbalanced change of IRF4 and IRF8 ratio in CpG ODN activated B cells

It is known that IRF4 and IRF8 are two major transcription factors that regulate the differentiation of activated B cells in an antagonistic manner, and their ratio affects the bifurcation of initial differentiation of B cells. To elucidate the possible mechanism that S2 tends to induce B cells to differentiate into IL-10-producing B cells despite negative regulation of B cell activation, we used the in vitro experimental platform of cultured mouse splenocytes to detect the expression of IRF4 and IRF8. We used flow cytometry to detect the changes in the percentage of CD19⁺ B cells and IRF4⁺/IRF8⁺ B cells in spleen cells, and analyzed the expression of IRF4 and IRF8 in B cells together or separately. It turned out that percentages of CD19⁺ B cells in CpG5805 and its combined S2 or MS19 groups were all similar to that in Med group, accounting for about 50–55% of spleen cells. The percentages of IRF4⁺ and IRF8⁺ B cells in the CpG5805-stimulated group were 3-fold and 2-fold of those in the Med group, respectively. However, S2 significantly suppressed this CpG5805-induced upregulation, whereas MS19 exhibited no inhibitory effect (Fig. 5a). Further analysis showed that the percentage of IRF8⁺ B cells was much higher than that of IRF4⁺ B cells, because it could be seen from the results that about half of IRF4⁺ and IRF4⁻ B cells were IRF8⁺ B cells, while only 10% or less of IRF8⁺ and IRF8⁻ B cells were IRF4⁺ B cells. CpG5805 could increase IRF8⁺ B cells in IRF4⁺ and IRF4⁻ B cells by 30%, respectively. S2 completely blocked the elevation effect of CpG5805 on IRF8⁺ B cells in IRF4⁻ B cells and IRF4⁺ B cells in IRF8⁺ B cells, but only reduced a half of the percentage of IRF8⁺ B cells in IRF4⁺ B cells increased by CpG5805 (Fig. 5b). This seems to indicate that S2 causes CpG ODN-induced B cells to have imbalanced changes in the ratio of IRF4 and IRF8.

Fig. 5.

Effect of S2 on CpG5805-induced expression of IRF4 and IRF8 in splenic B cells. Mouse splenocytes were stimulated with CpG5805 with or without S2/MS19 for 24 h, stained with fluorescence-labeled mAbs for surface CD19 and intracellular IRF4/IRF8, and then detected by flow cytometry. (a) The percentages of CD19⁺ and IRF4⁺/IRF8⁺ CD19⁺ cells in splenocytes. (b) Percentages of IRF8 and IRF4 expressing cells in IRF4⁺/IRF8⁺ or IRF4⁻/IRF8⁻ CD19⁺ B cells

S2 ODN as a decoy affected the nuclear translocations of IRF4 and IRF8 induced by CpG ODN

To find the possible reason for the change of IRF4 and IRF8 ratio in B cells induced by S2, considering that S2 was designed based on the consensus sequence recognized by the DNA binding domain (DBD) of IRFs, we adopted a pull-down assay to see if it could bind to IRF4 and IRF8. In this experiment, we still used MS19 as the control. Meanwhile, IRF5 was as another test object because MS19 is an inhibitory ODN that mainly targets IRF5 [33, 34]. We incubated Biotin-S2/MS19-bound Streptavidin-agarose beads with lysate of B cell line A20 cells followed by detection of IRF4, IRF5 and IRF8 by western blotting (Fig. 6a, left). In the Pull-down assay, when saturated lysate was given, both S2 and MS19 could bind to IRF4, IRF5 and IRF8 (Fig. 6a, middle), but when 1/10 diluted lysate was used, only S2 could bind to IRF4 and IRF8 while MS19 only bound to IRF5 (Fig. 6a, right), indicating that S2 had a high affinity with IRF4 and IRF8 while MS19 did not. This result suggests that S2 may act as a decoy ODN for either or both IRF4 and IRF8.

Fig. 6.

The pull-down assay for testing the binding ability of S2 to IRF4/5/8, and the effect of S2 on the phosphorylation and nuclear translocation of IRF4 and IRF8 in CpG5805-induced spleen cells. (a) Pull-down assay. The cell lysate was derived from B cell line A20 cells. Light arrow indicates the strip. (b) Effect of S2 on phosphorylation of IRF4 and IRF8 in spleen cells induced by CpG5805. (c) Effect of S2 on nuclear translocation of IRF4 and IRF8 in spleen cells induced by CpG5805. In (b) and (c), the result presented is one of the three experiments

It has been reported that decoy ODN binds to the target transcription factors and attenuates the gene expression they initiated at the pre-transcription level by affecting the translocation or activation of the transcription factors [37, 38]. To prove that S2 works as a decoy ODN, we used western blotting to detect the effects of S2 on the expression, phosphorylation and nuclear translocation of IRF4 and IRF8 in CpG5805-stimulated spleen cells. In this experiment, MS19 was still used as a control. The results showed that CpG5805 only slightly up-regulated the phosphorylated IRF8 in spleen cells but not for phosphorylated IRF4. The regulatory effects of S2 on both IRF4 and IRF8 phosphorylated in CpG5805 induced spleen cells were very weak, with the former slightly increasing and the latter slightly decreasing (Fig. 6b). In addition, S2 almost had no regulatory effect on CpG5805-induced IRF4 expression and nuclear translocation, but significantly reduced CpG5805-induced IRF8 expression and nuclear translocation (Fig. 6c). These results suggest that S2 may indeed act as a decoy to influence the activation, expression or nuclear translocation of IRF4 and IRF8, and seems to be more biased to affect the nuclear translocation of IRF8.

Discussion

The B cell response to the TLR9 agonist CpG ODN involves IRF4 and IRF8, which are antagonistic to each other in regulating the differentiation of activated B cells. The high levels of IRF4 were conducive to PC differentiation, while high levels of IRF8 can resistant to it. In this study, we demonstrated that S2 ODN negatively regulated CpG ODN-induced B cell activation by restoring the imbalanced IRF4 to IRF8 ratio, and favored the differentiation of some B cells into IL-10-producing Breg-like cells, rather than PCs. This suggests S2 ODN has the potential to treat disorders associated with B-cells abnormalities.

S2 is a single-stranded ODN containing the GAAA motif, which was designed based on the IRF consensus sequence [(GAAA(G/C) (T/C) GAAA] within the ISRE [5, 6]. In our study, we found that S2 could negatively regulate the proliferation and activation of splenic B cells stimulated by CpG ODN. This was reflected by the effective inhibition of CpG5805-induced upregulation of CD40 and TACI expression on these B cells, as well as the reduction in their sTLR9 levels, upon S2 treatment. Additionally, S2 successfully attenuated the elevated antibody responses induced by CpG5805 both in vitro and in vivo, alongside a reduction in IL-6 secretion from B cells. All these effects of S2 did not appear to be achieved through direct interference with the binding of CpG ODN to TLR9. This inference can be explained by the fact that S2 failed to inhibit the CpG5805-induced upregulation of Nf-κb and Prdm-1 mRNA levels in splenocytes. Nevertheless, this speculation requires further verification in future studies. TLR9 signaling involves the participation of several transcription factors, including IRF family members, NF-κB and Blimp-1. In detecting mRNA levels of Irf1-9 in spleen cells, we found that CpG5805 mainly upregulated the mRNA levels of Irf4, Irf5 and Irf8, while S2 downregulated their expression. This seems to make IRF4, IRF5, and IRF8 possible targets for S2, as they all belong to the IRF family and can bind to the IRF consensus sequence. However, TLR9-IRF5 signaling mainly induces the production of pro-/anti-inflammatory cytokines, while S2 significantly reduced CpG5805-induced IL-6 while upregulating IL-10. In addition, in the pull-down assay, S2 preferentially bound to IRF4 and IRF8 rather than IRF5. In contrast, MS19, an ODN previously shown to target IRF5 [33, 34], had obviously different effects from S2 on B cell responses and inflammatory cytokine. These results clearly indicate that S2 preferentially targets IRF4 and IRF8, rather than IRF5 or other IRF family members. S2 may function as a suppressive ODN that targets IRF4 and IRF8, possibly by acting as a decoy. It has been reported that suppressive ODNs typically interfere with immune signaling pathways [39]. Most known suppressive ODNs lack clearly defined cellular receptors. For example, A151, a well-characterized suppressive ODN based on mammalian telomeric repeats, has demonstrated protective effects in numerous murine models, of lupus, arthritis, uveitis, atopic dermatitis, infectious diseases (septic shock, fungal infections), and metabolic disorders [39]. Despite extensive study, its mechanism remains broadly attributed to immunosuppressive activity affecting STAT phosphorylation, given the central role of STAT proteins in immune cell maturation. Similarly, H154, a specific inhibitor of TLR9-mediated activation, reduces cytokine and antibody production and alleviates symptoms in models of myasthenia gravis (MG) and CpG ODN-induced cardiac dysfunction [40, 41]. Its mechanism is also limited to the understanding that it interferes with downstream TLR9 signaling without direct binding [40, 42]. Another example is MS19, an inhibitory ODN designed by our laboratory based on human microsatellite DNA. It reduced anti-ssDNA antibody levels and ameliorated lupus progression in a cGVHD model, and protected mice from virus-induced lung injury [36, 43]. Subsequent studies revealed that MS19 acts as a decoy by inhibiting IRF5 nuclear translocation [33, 34]. Notably, despite broad research on these inhibitory ODNs, their direct cellular receptors remain elusive. In the present study, we did not investigate the direct receptor for our newly designed S2 ODN, which will be a focus of future work. Although S2 had little effect on RF4 and IRF8 phosphorylation in CpG5805-stimulated spleen cells, we observed a slight increase in phosphorylated IRF4, consistent with previous report showing that murine gammaherpesvirus drives IL-10 production in B cells in a IRF4-dependent manner [44, 45]. S2 also down-regulated IRF8 expression and nuclear translocation. These findings suggest that S2 may act as a decoy ODN for IRF4 and IRF8 under TLR9 signaling, with a stronger bias toward IRF8. The unequal targeting may guide the differentiation direction of activated B cells.

B cells are typically activated encountering antigens or PAMPs and can differentiate into antibody-producing cells such as PCs. In this process, IRF4 and IRF8 regulate B cell fate in an antagonistic and concentration-dependent manner [15]. However, within this double negative feedback loop, interference may disrupt the orderly regulation by IRF4 and IRF8, potentially altering their normal stoichiometric ratio, thus changing the activation mode or differentiation direction of B cells. It has been reported that altered expression or imbalanced ratios of IRF4 and IRF8 in peripheral blood B cells is associated with clinical severity and frequency of circulating PCs in patients with MG, where IRF8 expression is negatively correlated with these clinical parameters in two MG subgroups [23]. In our study, we observed that S2 alters the direction of B cells differentiation. On the one hand, S2 reduced antibody production induced by CpG5805 or CpG5805 combined with HBV; on the other hand, it increased CpG5805-induced IL-10 production in B cells. This effect may be attributable to S2 modulating the ratio of IRF4 to IRF8, as we found that S2 completely inhibited the CpG5805-induced upregulation of IRF4⁺ B cells, but only partially reduced the upregulation of IRF8⁺ B cells. The increase in IL-10⁺ B cells induced by S2 may contribute to its ability to rescue mice from acute excessive inflammatory responses, given that IL-10⁺ B cells are critical for maintaining immune homeostasis and restraining immune responses in infection, cancer, and inflammation [30]. For instance, B cell-specific loss of IL-10 leads to increased pro-inflammatory cytokine expression, persistent leukocyte infiltration, and prolonged alveolar barrier damage in lipopolysaccharide (LPS)-induced ALI model mice [46]. Furthermore, S2 may process immunomodulatory potential for treating autoimmune diseases associated with TLR9 overactivation, due to its negative regulatory role in CpG ODN induced B cell responses. It has been reported that TLR9 signaling provides strong signals to B cells by initiating crucial molecules such as Blimp-1, TACI, NF-κB, and proinflammatory cytokines such as TNF-α and IL-6, thereby enhancing antigen responsiveness. This signaling may reduce B cell dependence on T cell help for responding to thymus-dependent antigens. Clinically, such reduced dependence may facilitate the development of B-cell-mediated autoimmune diseases like systemic lupus erythematosus (SLE), which is often associated with loss of T cell tolerance or excessive T cell help to B cells [47]. TLR9 ligands can further disrupt T cell tolerance [47]. Notably, TLR9 deficiency in B cells has been shown to promote the differentiation of IL-10⁺ Bregs and protect the hosts from developing Type 1 diabetes (T1D) [31]. In summary, the ability of S2 bias TLR9 signal-activated B cells toward differentiating into IL-10-producing Breg-like cells rather than PCs supports its potential as an immunomodulator for the treatment of excessive inflammatory response syndrome or certain autoimmune diseases.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

IRF

interferon regulatory factor

ODN

oligodeoxynucleotide

CpG ODN

CpG oligodeoxynucleotide

Bregs

regulatory B cells

PC

plasma cell

IL-10

Interleukin-10

TLR9

Toll-like receptor 9

sTLR9

surface Toll-like receptor 9

SIRS

systemic inflammatory response syndrome

Author contributions

Feiyu Lu was the main researcher for this study including the experiment design and manipulation, data analysis, and manuscript draft writing. Hong Wang, Kuo Qu, Mengru Zhu, Shengnan Wang, Tong Zhu have done some of the experiments covered in this article, including animal experiments, flow cytometry, western blotting, and immunofluorescence assay. Yongli Yu and Liying Wang provided research ideas, experiment design, writing and revising of the manuscript and funds.

Funding

This study is financially supported by the National Nature Scientific Foundation of China (No. 31670937).

Data availability

The authors confirm that the data supporting the findings of the present study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval

The mouse experiments were approved by the ethics committee of the College of Basic Medical Sciences, Jilin University (2024 − 295), and in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80 − 23) revised in 1996 and the guidelines of Jilin University.

Competing interests

The authors declare no competing interests associated with the manuscript.