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Streptococcal H2O2 inhibits IgE-triggered degranulation of RBL-2H3 mast cell/basophil cell line by inducing cell death

  • Nobuo Okahashi ,

    Roles Conceptualization, Formal analysis, Funding acquisition, Investigation, Writing – original draft

    okahashi@dent.osaka-u.ac.jp

    Affiliation Center for Frontier Oral Science, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan

  • Masanobu Nakata,

    Roles Formal analysis, Funding acquisition, Investigation, Writing – review & editing

    Affiliation Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan

  • Yujiro Hirose,

    Roles Investigation, Writing – review & editing

    Affiliation Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan

  • Hirobumi Morisaki,

    Roles Investigation, Writing – review & editing

    Affiliation Department of Oral Microbiology and Immunology, School of Dentistry, Showa University, Shinagawa, Tokyo, Japan

  • Hideo Kataoka,

    Roles Investigation, Writing – review & editing

    Affiliation Department of Oral Microbiology, Asahi University School of Dentistry, Mizuho, Gifu, Japan

  • Hirotaka Kuwata,

    Roles Formal analysis, Supervision, Writing – review & editing

    Affiliation Department of Oral Microbiology and Immunology, School of Dentistry, Showa University, Shinagawa, Tokyo, Japan

  • Shigetada Kawabata

    Roles Supervision, Writing – review & editing

    Affiliation Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan

Abstract

Mast cells and basophils are central players in allergic reactions triggered by immunoglobulin E (IgE). They have intracellular granules containing allergic mediators (e.g., histamine, serotonin, inflammatory cytokines, proteases and β-hexosaminidase), and stimulation by IgE-allergen complex leads to the release of such allergic mediators from the granules, that is, degranulation. Mast cells are residents of mucosal surfaces, including those of nasal and oral cavities, and play an important role in the innate defense system. Members of the mitis group streptococci such as Streptococcus oralis, are primary colonizers of the human oral cavity. They produce hydrogen peroxide (H2O2) as a by-product of sugar metabolism. In this study, we investigated the effects of streptococcal infection on RBL-2H3 mast cell/basophil cell line. Infection by oral streptococci did not induce degranulation of the cells. Stimulation of the RBL-2H3 cells with anti-dinitrophenol (DNP) IgE and DNP-conjugated human serum albumin triggers degranulation with the release of β-hexosaminidase. We found that S. oralis and other mitis group streptococci inhibited the IgE-triggered degranulation of RBL-2H3 cells. Since mitis group streptococci produce H2O2, we examined the effect of S. oralis mutant strain deficient in producing H2O2, and found that they lost the ability to suppress the degranulation. Moreover, H2O2 alone inhibited the IgE-induced degranulation. Subsequent analysis suggested that the inhibition of degranulation was related to the cytotoxicity of streptococcal H2O2. Activated RBL-2H3 cells produce interleukin-4 (IL-4); however, IL-4 production was not induced by streptococcal H2O2. Furthermore, an in vivo study using the murine pollen-induced allergic rhinitis model suggested that the streptococcal H2O2 reduces nasal allergic reaction. These findings reveal that H2O2 produced by oral mitis group streptococci inhibits IgE-stimulated degranulation by inducing cell death. Consequently, streptococcal H2O2 can be considered to modulate the allergic reaction in mucosal surfaces.

Introduction

Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii are oral mitis group streptococci, which are the most abundant inhabitants of the oral cavity and dental plaque [1, 2, 3, 4, 5]. They cause a variety of infectious complications such as bacteremia and infective endocarditis [5, 6, 7, 8, 9]. Streptococcus pneumoniae, an important pathogen that causes pneumonia, also belongs to the mitis group [8]. These mitis group streptococci produce hydrogen peroxide (H2O2) as a by-product of sugar metabolism [1, 3, 8, 10, 11, 12, 13, 14].

Mast cells and basophils are key effector cells in immunoglobulin E (IgE)-associated immune response, for example, anaphylaxis and allergic disorders such as allergic rhinitis and pollen-induced allergic rhinitis, that is, pollinosis [15, 16, 17, 18]. They have intracellular granules containing allergic mediators (e.g., histamine, serotonin, inflammatory cytokines, proteases and β-hexosaminidase) [15, 16, 18]. These cells constitutively express the IgE receptor (FcεRI) on their surface, and its aggregation by the IgE-allergen complex eventually leads to the release of such allergic mediators from the granules, that is, degranulation [15, 16, 17, 18]. In addition, they are associated with the innate immune response as well as autoimmune diseases, and also contribute to the initiation and progression of oral pathological conditions [15, 16, 18, 19, 20, 21]. Recently, as an allergen immunotherapy, sublingual immunotherapy has become a common treatment for pollinosis [22]. However, the influence of oral bacteria on mast cells in oral tissues and sublingual immunotherapy is unclear.

Previous studies [23, 24] have shown that infection with S. pneumoniae can activate mast cells. Other studies have reported that streptococcal toxins such as the pyrogenic exotoxin of Streptococcus pyogenes and hemolytic lipid toxin of Streptococcus agalactiae stimulate the degranulation of mast cells [25, 26]. These studies also suggest that modulation of mast cell function may contribute to the infection or colonization of the pathogenic streptococci.

We had previously reported that H2O2 produced by the oral mitis group streptococci induces the cell death of macrophages, epithelial cells and neutrophils, and its cytotoxicity is likely to contribute to the evasion of the streptococci from the host defense system [14, 27, 28, 29, 30]. Although our previous studies showed that streptococcal H2O2 is cytotoxic, the unique immune response of mast cells and basophils, i.e., IgE-induced degranulation, would raise another question. In this study, we investigated whether H2O2 produced by the oral mitis group streptococci is implicated in the allergic function.

Materials and methods

Ethics statement

The mouse experiments were performed with the approval of the animal care committee of the Osaka University Graduate School of Dentistry (No, 29-009-0). All experiments were performed according to the guidelines for animal treatment of the committee.

Chemicals and reagents

Brain heart infusion (BHI) broth was purchased from Becton Dickinson (Sparks, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM) and other cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA). Mouse anti-dinitrophenol (DNP) IgE monoclonal antibody, DNP-conjugated human serum albumin (HSA), p-nitrophenyl-N-acetyl-β-d-glucosaminide (PNAG), phorbol 12-myristate 13-acetate (PMA), ionomycin, trypan blue, staurosporine, and catalase were obtained from Sigma Aldrich (St. Louis, MO, USA). Astra Blue dye solution was purchased from ScyTek (Logan UT, USA). LysoTracker Red and SYBR Green II were purchased from Molecular Probes (Eugene, OR, USA) and TaKaRa Bio (Otsu, Japan), respectively. 4,6-Diamidino-2-phenylindole (DAPI) and AlexaFluor 594-conjugated phalloidin were obtained from Dojindo Molecular Technologies (Kumamoto, Japan) and Molecular Probes, respectively. Rabbit anti-β-actin antibody and horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody were purchased from Cell Signaling (Danvers, MA, USA). Other commonly used reagents were purchased from Nakalai Tesque (Kyoto, Japan) and Sigma-Aldrich.

Bacterial strains and culture conditions

S. oralis ATCC 35037, a type strain originally isolated from the human mouth [2], was obtained from the Japan Collection of Microorganisms at the RIKEN BioResource Center (Tsukuba, Japan). The spxB-deletion mutant, spxB KO (deficient for H2O2 production), was generated from S. oralis ATCC 35037 wild type (WT), as described previously [14].

Streptococcus salivarius HHT and S. gordonii ATCC 10558 were selected from the stock culture collection at the Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry (Osaka, Japan). S. salivarius does not produce detectable H2O2 [1, 3], and S. gordonii is a member of the oral mitis group of streptococci [3, 10, 13]. These bacteria were cultured in BHI broth.

Cell culture

The rat mast cell/basophil cell line RBL-2H3 (JCRB0023) [31] was obtained from the JCRB Cell Bank (Ibaraki-Osaka, Japan). The cell line has been widely used as a mast cell line in the IgE-stimulated degranulation studies, however, recent studies suggested that this cell line share some characteristics with basophils [32, 33]. The cells were cultured in DMEM supplemented with 5% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C in a 5% CO2 atmosphere. For the degranulation assay (see below), the cells were cultured in 5% FBS DMEM containing no phenol red.

Effects of streptococcal infection and H2O2 on degranulation of RBL-2H3 cells

The RBL-2H3 cells (5 × 105 cells) in the 24 well plates were infected with the streptococcal strains at a multiplicity of infection (MOI) of 200, or treated with H2O2 (2 mM) for 3 h. A mixture of PMA (10 nM) and ionomycin (1 μM) (PMA + ionomycin) was used as the positive control for degranulation [34]. The supernatants were then centrifuged at 10,000 × g for 10 min to remove the detached cells and bacteria. The release of allergic mediators by degranulation was monitored using the β-hexosaminidase assay [35]. The clarified supernatants (50 μL) were mixed with 100 μL of substrate solution (2 mM PNAG in 0.1 M sodium citrate buffer, pH 4.5) in 96 well microtiter plates, and the mixture was incubated for 1 h at 37°C. The reaction was terminated by adding 50 μL of 2 M glycine buffer (pH 10). Absorbance at 405 nm was measured using a Multiskan FC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The total β-hexosaminidase activity was also measured using a whole cell lysate of the RBL-2H3 cells lysed with 0.1% Triton X100. The released β-hexosaminidase activity was expressed in percentage by using the following equation: where A total is the absorbance of the reaction with the whole cell lysate, A sample is the absorbance of the samples, and A blank is the absorbance of the blank reaction mixture.

IgE-antigen complex-triggered degranulation of RBL-2H3 cells

The RBL-2H3 cells (5 × 105 cells) in 24 well plates were sensitized with mouse monoclonal anti-DNP IgE antibody (50 ng/mL) for 2 h. The cells were then washed with phosphate buffered saline (PBS; pH 7.2), cultured in a new medium containing no phenol red and antibiotics, and infected with streptococcal strains at an MOI of 10, 50 or 200, or treated with H2O2 (0.1, 0.5 or 2 mM) for 3 h. Then, the cells were stimulated for 30 min with DNP-conjugated HSA (25 ng/mL). The culture supernatants were centrifuged at 10 000 × g for 10 min to remove detached cells and bacteria. The hexosaminidase activity in the culture supernatants was determined as described above.

Cell death of RBL-2H3 cells

The RBL-2H3 cells (2 × 105 cells in 5% FBS DMEM) were infected with the viable streptococcal strains at an MOI of 200, in the absence of antibiotics, for 3 h. The culture medium was changed to a fresh medium containing antibiotics, and cultured for an additional 18 h. The cells were then stained with 0.2% trypan blue in PBS. After incubation at room temperature for 10 min, the numbers of viable and dead cells were counted using a microscope (Nikon TMS-F; Nikon, Tokyo, Japan). Because the dead RBL-2H3 cells were easily detached from the bottom of the culture plates, cells that disappeared during the washing and staining steps were considered to be dead (see S1 Fig). Cell death induced by H2O2 (2 mM) or PMA (10 nM) + ionomycin (1 μM) was determined similarly. To evaluate the dose-dependent effect, the cells were infected with viable S. oralis WT (MOI = 10, 50 or 200), or treated with H2O2 (0.1, 0.5 or 2 mM).

The effect of catalase was also investigated. Prior to infection, 10, 50 or 200 U/mL of catalase was added to the culture of RBL-2H3 cells, and the cells were then infected with viable S. oralis WT (MOI = 200) for 3 h. The cells were washed with PBS and cultured in fresh medium containing catalase and antibiotics for 18 h. The viability was determined as described above.

Astra Blue staining and acidic lysosome staining

The RBL-2H3 cells were cultured on Cell Desk LF (Sumitomo Bakelite, Tokyo, Japan) in 24 well culture plates and exposed to S. oralis WT, spxB KO (MOI = 200), H2O2 (2 mM) or PMA + ionomycin for 3 h, changed to fresh medium containing antibiotics, and cultured for an additional 3 h (total 6 h). The cells were fixed overnight with 10% formaldehyde at 4℃ and stained with Astra Blue dye solution. The Astra Blue dye stains heparin in the granules of mast cells as blue [36].

The RBL-2H3 cells were cultured as described above, and the viable cells were stained with LysoTracker Red probe (50 nM) and SYBR Green II (1:2000 dilution) in culture medium for 15 min, washed with PBS, and observed using a Carl Zeiss Axioplan 2 fluorescent microscope system (Carl Zeiss, Oberkochen, Germany). LysoTracker Red is an acidotropic red fluorescent probe that accumulates in the acidic lysosomes. SYBR Green is a DNA-binding dyes that stains the nuclei.

Fluorescence staining of actin

The RBL-2H3 cells were cultured on Cell Desk LF and exposed to S. oralis WT, spxB KO (MOI = 200), H2O2 (2 mM), or PMA + ionomycin for 3 h, washed with PBS, and cultured for an additional 3 h (total 6 h) in fresh medium containing antibiotics. The cells were fixed with 10% formaldehyde, followed by permeabilization with 0.2% Triton X-100. The DNA and actin filaments were labeled with DAPI (1 μg/mL) and AlexaFluor 594-conjugated phalloidin (1:200 dilution) in PBS for 15 min. After washing with PBS, the cell fluorescence was observed.

Annexin V staining

The RBL-2H3 cells were cultured on Cell Desk LF and exposed to S. oralis WT, spxB KO (MOI = 200), H2O2 (2 mM), or PMA + ionomycin for 3 h, washed with PBS, and cultured for an additional 3 h (total 6 h) in fresh medium containing antibiotics. The cells were stained using fluoresceinisothiocyanate isomer (FITC)-Annexin V staining kit (MBL, Nagoya, Japan), according to the protocol of the manufacturer, and the fluorescence was observed using a fluorescent microscope. Differential interference contrast (DIC) images were taken in the same fields. As a positive control for apoptosis, the cells were stimulated by staurosporine (10 μM) [37] for 1 and 3 h. FITC-Annexin V (green fluorescence) binds to the apoptotic cells, and PI (red fluorescence) stains dead cells.

Lactate dehydrogenase (LDH) assay

The RBL-2H3 cells (5 × 105 cells) in 24 well plates were infected with viable S. oralis WT or spxB KO strains (MOI; 200) in the absence of antibiotics for 3 h. In order to stop the bacterial growth, antibiotics (penicillin [100 U/mL], and streptomycin [100 μg/mL]) were added, and the cells were cultured for additional 3 h (total 6 h). Cells were also treated with H2O2 (2 mM) or PMA + ionomycin. The supernatants were then centrifuged at 10,000 × g for 10 min to remove detached cells and bacteria. The LDH activity in the culture supernatants was measured using Cytotoxicity detection LDH kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions.

Interleukin-4 (IL-4) assay

Culture supernatants of the RBL-2H3 cells exposed to viable S. oralis WT, spxB KO strains (MOI 200), H2O2 (2 mM) or PMA + ionomycin were assayed for IL-4 by using an enzyme-linked immunosorbent assay (ELISA) kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The cells were also sensitized with mouse anti-DNP IgE monoclonal antibody, and then with DNP-conjugated HSA, as described above.

Effects of nasal inoculation of S. oralis and H2O2 on pollen-induced murine allergic rhinitis

The mouse experiments were performed with the approval of the animal care committee of the Osaka University Graduate School of Dentistry (No, 29-009-0). Female ddY mice (4-week-old) were purchased from Japan SLC (Hamamatsu, Japan) and fed a standard rodent diet with water ad libitum and maintained at 22–24°C in the animal facility at the Osaka University Graduate School of Dentistry under a 12 h/12 h light/dark cycle. To exclude the effect of the genetic background, we used the ddY inbred mice in this study. Allergic rhinitis was induced in the mice by sensitization to Japanese cedar pollen (Wako Pure Chemicals; Osaka, Japan), according to the protocols for ragweed pollen-induced allergic murine rhinitis (Fig 8A) [38, 39]. For sensitization, the mice were intraperitoneally immunized with cedar pollen (0.1 mg) with alum adjuvant (1 mg) in 200 μL of PBS on days 0, 7, and 14. The mice were further immunized by repeated intranasal administration of cedar pollen (0.5 mg/20 μL) on days 17, 18, 21–25, 31, 32, and 35–39. As a negative control, the mice were intraperitoneally administered with the alum adjuvant alone, following intranasal administration of PBS. The mice were examined for sensitization at day 42, and fully sensitized mice were subjected to further experiments. To examine the effects of S. oralis infection or H2O2 administration, the pollen-sensitized mice (4 mice per each group) were intranasally inoculated with bacterial cultures (5 × 107 colony forming unit [CFU] in DMEM, 10 μL × 5 times) of S. oralis WT or spxB KO, or H2O2 (10 mM in DMEM, 10 μL × 5 times). The mice were challenged by intranasal inoculation of pollen (0.5 mg/20 μL) after 6 h, and the frequency of sneezing and scraping for 5 min was counted (Fig 8). The nasal inoculum (5 × 107 CFU) was relevant to the murine nasal infection of S. pneumoniae [40].

Statistical analysis

The statistical analyses were performed using QuickCalcs software (GraphPad Software, La Jolla, CA, USA) and Ekuseru Toukei (Social Survey Research Information, Tokyo, Japan). The statistical differences were examined using independent Student’s t-test. We also compared multiple groups using a two-tailed one-way analysis of variance (ANOVA) with Dunnett’s test. A confidence interval with a p value of < 0.05 was considered to be significant.

Results

Streptococcal infection did not induce degranulation of RBL-2H3 cells

The rat mast cell/basophil cell line RBL-2H3 [31] was used to assess the in vitro effects of streptococcal infection on degranulation through a β-hexosaminidase assay [34, 35]. At first, we investigated the direct effect of streptococcal infections on RBL-2H3 cells. Viable S. oralis WT, S. oralis spxB KO (deficient for H2O2 production), S. gordonii, or S. salivarius at an MOI of 200 did not induce the release of β-hexosaminidase. Stimulation by PMA + ionomycin induced the release of β-hexosaminidase, as reported previously [34]. H2O2 (2 mM) was included in this assay, because the mitis group streptococci produce 1–3 mM of H2O2 [14, 29]. H2O2 did not elicit the release of β-hexosaminidase. These results suggest that infection with oral streptococci or exposure to H2O2 did not stimulate the degranulation of RBL-2H3 cells (Fig 1).

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Fig 1. Streptococcal infection did not induce degranulation of RBL-2H3 cells.

RBL-2H3 cells were infected with streptococcal strains at an MOI of 200 or treated with H2O2 (2 mM) for 3 h. A mixture of PMA (10 nM) and ionomycin (1 μM) was used as the positive control. The culture supernatants were then centrifuged to remove the detached cells and bacteria. The released β-hexosaminidase activity was determined using the PNAG substrate. The activity in the whole cell lysate was referred to as 100%. The data are shown as mean ± SD values of triplicate samples. *p < 0.05 as compared with the untreated control (None).

https://doi.org/10.1371/journal.pone.0231101.g001

Effects of streptococcal infection on IgE-induced RBL-2H3 cell degradation

Next, we investigated the effects of streptococcal infection on IgE-induced degranulation of the RBL-2H3 cells. The cells were sensitized with mouse anti-DNP IgE, and then exposed to streptococcal strains or H2O2. Then, the cells were stimulated with DNP, and the release of β-hexosaminidase was measured. Viable S. oralis WT or S. gordonii at an MOI of 200 inhibited the release of β-hexosaminidase from the RBL-2H3 cells stimulated with the IgE-antigen complex (Fig 2A). S. oralis spxB KO or S. salivarius, which does not produce H2O2, did not inhibit the IgE-triggered β-hexosaminidase release. H2O2 alone was sufficient to inhibit the degranulation (Fig 2A). These results suggest that H2O2 produced by oral mitis group streptococci inhibits IgE-stimulated degranulation.

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Fig 2. Inhibition of IgE-induced degranulation by streptococcal infection and H2O2.

(A) RBL-2H3 cells were sensitized with mouse anti-DNP IgE monoclonal antibody, and then infected with streptococcal strains at an MOI of 200 or treated with H2O2 (2 mM) for 3 h. Then, the cells were stimulated for 30 min with DNP-conjugated HSA. The β-hexosaminidase activity in the supernatants was determined using the PNAG substrate. (B) RBL-2H3 cells sensitized with mouse anti-DNP IgE monoclonal antibody were infected with S. oralis WT at MOI of 10, 50, or 200 or treated with H2O2 (0.1, 0.5, or 2 mM). Then, the cells were stimulated for 30 min with DNP-conjugated HSA. The β-hexosaminidase activity in the supernatants was determined using the PNAG substrate. The activity in whole cell lysate was referred to be 100%. The data are shown as mean ± SD values of triplicate samples. *p < 0.05 as compared with the untreated IgE-stimulated control (anti-IgE + DNP, None).

https://doi.org/10.1371/journal.pone.0231101.g002

The inhibitory effect of streptococcal H2O2 was dose-dependent (Fig 2B). The release of β-hexosaminidase decreased with increased infection of S. oralis WT, or increased concentration of H2O2 (Fig 2B).

Streptococcal H2O2 induced the cell death of RBL-2H3 cells

We had previously reported that infection with oral mitis group streptococci induces the death of macrophages, epithelial cells, and neutrophils with streptococcal H2O2 contributing to the cell death [14, 28, 29]. Therefore, we next examined whether H2O2 produced by the oral mitis group streptococci is cytotoxic to RBL-2H3 cells. The RBL-2H3 cells were exposed to viable oral streptococcal strains or H2O2, and the cells were stained with trypan blue to determine their viability (Fig 3A). Cytotoxicity of PMA + ionomycin, which induce degranulation (Fig 1), was also examined. Viable S. oralis or S. gordonii induced the cell death of the RBL-2H3 cells. Exposure to spxB KO or S. salivarius had little effect on the cellular viability. H2O2 was cytotoxic, and PMA + ionomycin showed moderate cytotoxicity (Fig 3A). Since infection with H2O2-producing streptococci or exposure to H2O2 resulted in the detachment of the cells from the bottom of the culture plate, we also examined the viability of the detached cells, and found that they were also dead (S1 Fig).

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Fig 3. Streptococcal infection and H2O2 induce RBL-2H3 cell death.

(A) RBL-2H3 cells were infected with viable streptococcal strains at an MOI of 200, in the absence of antibiotics, for 3 h. The cells were then cultured for 18 h in fresh medium containing antibiotics. Viability of the cells was determined using the trypan blue dye exclusion method. Viability of the cells treated with H2O2 (2 mM) or PMA (10 nM) + ionomycin (1 μM) was also determined. (B) To evaluate the dose dependent effect, the cells were infected with viable S. oralis WT at an MOI of 10, 50 or 200, or treated with H2O2 (0.1, 0.5 or 2 mM), and the viability was determined using the trypan blue dye exclusion method. (C) Involvement of streptococcal H2O2 was studied using catalase. Prior to infection, 10, 50 or 200 U/mL of catalase was added to the culture of RBL-2H3 cells, and the cells were then infected with viable S. oralis WT (MOI = 200). Viability was determined as described above. The data are shown as mean ± SD values of triplicate samples. *p < 0.05 as compared with the untreated control (None).

https://doi.org/10.1371/journal.pone.0231101.g003

The dose-dependent effects of streptococcal infection and H2O2 on cellular viability were also examined. As shown in Fig 3B, streptococcal infection with MOI of more than 50 and H2O2 concentration of more than 0.5 mM were found to induce cell death of RBL-2H3 cells. Based on the dose-dependency of cytotoxicity, we additionally examined the effects of sub-cytotoxic doses of S. oralis WT (MOI = 1, 2 and 5) and H2O2 (0.01, 0.02, 0.05 mM) on the IgE-induced degranulation (S2 Fig). However, these low doses of S. oralis WT or H2O2 showed no significant effect (S2 Fig).

To examine the contribution of H2O2, we investigated the effect of catalase, an H2O2-decomposing enzyme, on S. oralis-induced cell death. Exogenously added catalase reduced death in the RBL-2H3 cells infected with S. oralis WT (Fig 3C).

Staining for granules and lysosomes of RBL-2H3 cells

The granules of RBL-2H3 cells exposed to S. oralis WT, spxB KO or H2O2, as well as PMA + ionomycin were visualized with Astra Blue staining [36]. Many of the intact cells that contained granules were stained blue (Fig 4A, None), whereas the cells treated with PMA + ionomycin showed reduced staining because of degranulation (Fig 4A, P+I). S. oralis spxB KO did not trigger degranulation (Fig 4A, KO). The cells treated with S. oralis WT or H2O2 were stained blue (Fig 4A). Because the dead cells were detached from the bottom of the culture plate, the number of visible cells decreased in the cell cultures treated with S. oralis WT or H2O2.

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Fig 4. Astra Blue and acidic lysosome staining.

(A) RBL-2H3 cells cultured on Cell Desk LF were exposed to S. oralis WT, spxB KO (MOI = 200), H2O2 (2 mM) or PMA (10 nM) + ionomycin (1 μM) for 3 h, and cultured for an additional 3 h (total 6 h) in fresh medium containing antibiotics. The cells were fixed with 10% formaldehyde, and stained with Astra Blue dye. (B) The viable cells were stained with LysoTracker Red probe and SYBR Green II, and observed using a fluorescent microscope. LysoTracker Red is a probe that accumulates to acidic lysosomes. SYBR Green stains the nuclei. Bar = 10 μm.

https://doi.org/10.1371/journal.pone.0231101.g004

Our previous study suggests that lysosomal damage contributes to macrophage cell death induced by H2O2 [41]. Therefore, the RBL-2H3 cells were stained with LysoTracker, an acidotropic fluorescent probe (Fig 4B). Although a limited number of LysoTracker-positive lysosomes was detectable in the RBL-2H3 cells (None), the fluorescent intensity decreased by infection with S. oralis WT or exposure to H2O2, suggesting deacidification of the lysosomes occurred during the bacterial infection or exposure to H2O2. In addition, lysosomal damage was not observed in S. oralis spxB KO-infected cells.

Fluorescence staining of actin of RBL-2H3 cells

We found that the dead RBL-2H3 cells easily detached from the bottom of the culture plate. Therefore, we examined the actin filaments in the RBL-2H3 cells treated with S. oralis WT, spxB KO or H2O2 (Fig 5). Immunofluorescence staining of actin showed decreased actin filaments in the RBL-2H3 cells exposed to S. oralis WT or H2O2, whereas S. oralis spxB KO did not induce such morphological change (Fig 5).

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Fig 5. Fluorescence staining of actin.

RBL-2H3 cells on the Cell Desk were exposed to S. oralis WT, spxB KO (MOI = 200), H2O2 (2 mM) or PMA (10 nM) + ionomycin (1 μM) for 3 h (upper images), and cultured for an additional 3 h (total 6 h; lower images) in fresh medium containing antibiotics. The cells were fixed with 10% formaldehyde, followed by permeabilization with 0.2% Triton X-100. The DNA and actin filaments were labeled with DAPI and AlexaFluor 594-conjugated phalloidin. Bar = 10 μm.

https://doi.org/10.1371/journal.pone.0231101.g005

Annexin V staining and release of LDH

To evaluate whether streptococcal infection or H2O2 exposure induces necrotic or apoptotic cell death, FITC-Annexin V staining and LDH release were investigated (Fig 6). Annexin V is known to bind to apoptotic cells, and thus, the apoptotic cells show green fluorescence. PI (red fluorescence) stains all dead cells. Cells treated with staurosporine, an apoptosis inducer [37], were stained by both Annexin V and PI. However, Annexin V did not bind to the RBL-2H3 cells exposed to S. oralis WT or H2O2 (Fig 6A). These cells were stained by PI, indicating that they were dead. Infection by S. oralis spxB KO or exposure to PMA + ionomycin did not induce cell death (Fig 6A). These results suggested that the cell death induced by strepotococcal H2O2 was not apoptosis.

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Fig 6. Annexin V fluorescent staining and LDH release.

(A) The RBL-2H3 cells were cultured on Cell Desk LF and exposed to S. oralis WT (MOI = 200), H2O2 (2 mM), or PMA (10 nM) + ionomycin (1 μM) for 3 h, and were stained using FITC-Annexin V staining kit. Staurosporine (10 μM) was used as a positive control for apoptosis. FITC-Annexin V (green fluorescence) binds to the apoptotic cells, and PI (red fluorescence) stains dead cells (lower images). Differential interference contrast (DIC) images were taken in the same fields (upper images). Bar = 20 μm. (B) RBL-2H3 cells (5 × 105 cells) in 24 well plates were infected with viable S. oralis WT (MOI = 200) in the absence of antibiotics for 3 h. Then, the infected cells were cultured in the presence of antibiotics for an additional 3 h (total 6 h). The cells were also treated with H2O2 (2 mM), PMA (10 nM) + ionomycin (1 μM) or staurosporine (10 μM). The culture supernatants were then centrifuged to remove the detached cells and bacteria. LDH in the culture supernatants was measured using an LDH activity assay kit according to the manufacturer’s instructions.

https://doi.org/10.1371/journal.pone.0231101.g006

LDH release from dead cells is used as an indicator of necrotic cell death. Cells undergoing apoptotic death do not release LDH in their early cell death stage, because the cell membranes are intact. At the early stage (3 h), infection by S. oralis WT, or exposure to H2O2 did not stimulate LDH release from the RBL-2H3 cells. However, increased LDH release at 6 h after exposure to S. oralis WT or H2O2 was observed (Fig 6B, left). Cells treated with staurosporine gradually released LDH in the culture medium (Fig 6B, right).

IL-4 release from RBL-2H3 cells

Activated mast cells and basophils are reported to produce IL-4 [15, 16, 18, 21]. Thus, IL-4 in the culture medium of the treated cells was also measured (Fig 7). Infection by S. oralis WT or spxB KO, or exposure to H2O2 did not stimulate IL-4 production from the RBL-2H3 cells, whereas PMA + ionomycin induced its production (Fig 7, left). As well as PMA + ionomycin, activation by IgE-antigen complex stimulated IL-4 production (Fig 7, right). We also found that sub-cytotoxic doses of S. oralis WT or H2O2 showed no significant effect on Il-4 production from RBL-2H3 cells (S3 Fig).

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Fig 7. IL-4 production from RBL-2H3 cells.

RBL-2H3 cells (5 × 105 cells) in 24 well plates were infected with viable S. oralis WT (MOI = 200) in the absence of antibiotics for 3 h. Then, the infected cells were cultured in the presence of antibiotics for an additional 3 h (total 6 h). The cells were also treated with H2O2 (2 mM) or PMA (10 nM) + ionomycin (1 μM). The cells were also sensitized with mouse anti-DNP IgE monoclonal antibody, and then with DNP-conjugated HSA. The culture supernatants were then centrifuged to remove the detached cells and bacteria. The amount of IL-4 in the culture supernatants was measured using an ELISA kit. The data are shown as mean ± SD values of triplicate samples. *p < 0.05 as compared with the untreated control (None).

https://doi.org/10.1371/journal.pone.0231101.g007

In vivo study of the effects of nasal inoculation of S. oralis and H2O2 on pollen-induced murine allergic rhinitis

To confirm the effect of streptococcal H2O2 on in vivo allergic reaction, a murine pollen-induced allergic rhinitis model [38, 39] was used. Repeated immunization of mice with cedar pollen successfully induced allergic rhinitis (Fig 8). Then, the sensitized mice were intranasally inoculated with S. oralis culture or H2O2. As compared with positive control mice and mice infected with spxB KO strain, mice either infected by S. oralis WT or exposed to H2O2 exhibited the reduced number of sneezes and scraping after the pollen challenge, suggesting that the inhibition of the allergic reaction is mediated by H2O2 (Fig 8). However, underlying mechanisms remain to be conclusively defined.

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Fig 8. Effect of nasal inoculation of S. oralis and H2O2 on pollen-induced murine allergic rhinitis.

(A) The mice were intraperitoneally immunized with cedar pollen (0.1 mg) with alum adjuvant (1 mg) in 200 μL of PBS three times, and further immunized by repeated intranasal administration of cedar pollen (0.5 mg/20 μL). The mice were examined for sensitization on day 42, and the fully sensitized mice were subjected to further experiments. The pollen-sensitized mice (4 mice per each group) were intranasally inoculated with bacterial cultures (5 × 107 CFU in DMEM, 10 μL × 5 times) of S. oralis WT, spxB KO, or H2O2 (10 mM in DMEM, 10 μL × 5 times). The mice were challenged by intranasal inoculation of pollen (0.5 mg/20 μL) after 6 h, and the frequency of sneezing (B) and scraping (C) for 5 min was counted. The data are shown as mean ± SD values of 4 mice. *p < 0.05 as compared with the positive control (None, pollen challenge alone).

https://doi.org/10.1371/journal.pone.0231101.g008

Discussion

This study reveals that H2O2 produced by oral mitis group streptococci inhibits degranulation of the RBL-2H3 mast cells/basophils stimulated by the IgE-allergen complex. The cytotoxicity of the streptococcal H2O2 contributes to the inhibition of degranulation. The results are summarized in Fig 9.

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Fig 9. Summary of this study.

(A) Under normal allergy conditions, allergen-binding IgE induces degranulation from mast cells. (B) Infection by H2O2-producing oral mitis group streptococci causes the death of mast cells. The IgE-antigen complex cannot induce the degranulation of the streptococci-infected mast cells.

https://doi.org/10.1371/journal.pone.0231101.g009

In our previous studies, we have shown that H2O2 produced by oral mitis group streptococci is cytotoxic to host innate immune cells such as macrophages, neutrophils, and epithelial cells [14, 27, 28, 29]. Moreover, we recently found that streptococcal H2O2 induces a stress response in macrophages [30]. Such stress responses could contribute to the suppression of degranulation in mast cells/basophils. Other studies have also reported the cytotoxicity and pathogenicity of streptococcus-derived H2O2 [42, 43, 44, 45, 46]. These findings suggest that streptococcal H2O2 has beneficial effects on streptococcal colonization of the mucosal surfaces and even on infections leading to cardiovascular diseases.

Infection with S. pneumoniae is reported to activate mast cells [23, 24]. Fritscher et al. [24] showed that the activation of mast cells is dependent on a pore-forming cytolysin named pneumolysin [47]. Other studies have shown that pyrogenic exotoxin of S. pyogenes and hemolytic lipid toxin of S. agalactiae stimulate the degranulation of mast cells [25, 26]. These studies also suggest that pathogenic streptococci modulate mast cell function to evade the host immune response.

Regarding to the effect of H2O2 on the mast cells, Peden et al. [48] reported that H2O2 (0.2–2 mM) inhibits cell proliferation and IgE-induced degranulation of the RBL-2H3 cells. The concentration of H2O2 in their study was similar to the concentration in the culture supernatants of S. oralis WT (1–3 mM) [14, 29]. Other studies also have reported that exogenous H2O2 impairs the degranulation of mast cells [49, 50]. In contrast, intracellular reactive oxygen species produced by mitochondria have been reported to contribute to the degranulation and cytokine production of mast cells [51]. These studies do not address the cytotoxicity of H2O2 against mast cells, whereas our present study clearly demonstrated that the cytotoxicity of streptococcal H2O2 plays an important role in the inhibition of the IgE-induced degranulation.

To test the potential effects of streptococcal H2O2 on in vivo allergic reaction, we used a murine pollen-induced allergic rhinitis model (Fig 8). Pretreatment with streptococcal culture or H2O2 resulted in a significant reduction of the allergic reaction, such as sneezing and scraping, after the pollen challenge (Fig 8). Thus, streptococcal H2O2 may reduce the allergic reaction through its suppressive effect on mast cells and basophils. Possibly, suppression of other immune cells such as macrophages [41] and neutrophils [29] is involved in the reduction of the allergic reaction. It should be noted that due to non-negligible variation in methods of in vivo pollen-induced rhinitis model conducted by each group [38, 39], it is difficult to compare our results to those of previous studies. For example, Haenuki et al. [38] sensitized mice of C57BL/6 background with ragweed pollen, while Kato et al. [39] sensitized BALB/c mice. To exclude the effect of the genetic background, we used ddY inbred mice in this study, and the level of the allergic response such as sneezing is similar to those of their results. Although S. oralis is not considered to be major inhabitant of the nasal cavity, a study using human specimen shows that the Streptococcus genus was represented in nasal cavity and S. oralis/Streptococcus mitis (these two species are very similar) was most prevalent members (17.6%) [52]. S. pneumoniae is one of the residents of the nasal cavity [53, 54], and therefore, pneumococcal H2O2 is likely to damage the immune response in the nasal cavity [42, 43, 44]. To appraise the contribution of nasal and/or oral mitis group streptococci to the inhibition of the allergic response, more detailed analysis including human clinical studies will be required.

Recent studies have revealed that mast cells and basophils control the innate immune response in various ways. IgE-induced immune response and degranulation from mast cells and basophils are considered to be host defense responses against parasites [15, 16, 18, 19, 21, 55]. In this study, we found that H2O2 produced by mitis group streptococci inhibits the IgE-induced degranulation from RBL-2H3 cells. Moreover, streptococcal H2O2 does not stimulate the production of IL-4 from the cells. Such suppressive effect of the innate immune response is considered to be an evasion strategy of streptococci to escape from the host immune response, thereby supporting streptococcal colonization at the mucosal surface of the oral cavity [30, 55]. In this regard, previous studies have suggested that inhibition of degranulation of mast cells prevents both inflammatory and allergic responses [56, 57]. Interestingly, commensal bacteria are necessary for the maintenance of a healthy mucosal immune system. Commensal Clostridia can contribute to immune homeostasis in the intestine by inducing the differentiation of regulatory T cells through synthesis of short-chain fatty acids such as butyrate [58]. On the other hand, a recent study suggested that microbial oxygen respiration contributes to intestinal inflammation [59]. H2O2 from oral streptococcus may have such potential immunomodulatory effect on the innate immune system in the oral cavity.

We had previously reported that streptococcal H2O2 induces the death of macrophages, and dysfunction of lysosomes contributes to cell death [41]. Lysosomes are organelles filled with “cytotoxic” hydrolytic enzymes, including proteases, and their dysfunction is considered to induce cell death [60, 61]. Therefore, we examined the effect of streptococcal H2O2 on lysosomal integrity of RBL-2H3 cells (Fig 4). LysoTracker fluorescent staining demonstrated that the streptococcal H2O2 elicited a reduction in the acidic lysosomal environment. Effect of deferoxamine, which is an iron chelator and reduces the production of peroxide radicals from H2O2 within lysosomes [62, 63], also suggests the involvement of lysosomal dysfunction in the cell death of mast cells (S4 Fig).

Braun et al. [43] and Rai et al. [45] have revealed that pneumococcal H2O2 induces apoptosis in microglia and lung cells, respectively. However, in our study, the involvement of apoptotic event in H2O2-induced RBL-2H3 cell death was not evident (Fig 6). The dead RBL-2H3 cells were Annexin V-negative, suggesting that their cell death is not apoptosis. It should be noted that the H2O2-induced cell death was not accompanied with degranulation, thereby inducing limited inflammatory response. In this study, we used only one cell line, RBL-2H3, and therefore our findings have to be confirmed using other cell lines or primary mast cells in future studies.

In summary, our results reveal that H2O2 produced by oral mitis group streptococci inhibit the degranulation of mast cells/basophils through the induction of cell death, suggesting that H2O2 plays a significant role in the modulation of the innate immune response induced by mast cells in the mucosal surface. Effects of streptococcal H2O2 on the local allergic response such as pollen-induced rhinitis or metal allergy will be of special interest for future studies.

Supporting information

S1 Fig. Trypan blue staining of detached RBL-2H3 cells.

https://doi.org/10.1371/journal.pone.0231101.s001

(TIFF)

S2 Fig. Effects of sub-cytotoxic doses of S. oralis and H2O2 on IgE-induced degranulation of RBL-2H3 cells.

https://doi.org/10.1371/journal.pone.0231101.s002

(TIFF)

S3 Fig. Effects of sub-cytotoxic doses of S. oralis and H2O2 on IL-4 production of RBL-2H3 cells.

https://doi.org/10.1371/journal.pone.0231101.s003

(TIFF)

S4 Fig. Effect of deferoxamine (DFO) on the cell death of RBL-2H3 cells.

https://doi.org/10.1371/journal.pone.0231101.s004

(TIFF)

S1 Appendix. Supplementary Materials and Methods, Results and Discussion for S4 Fig.

https://doi.org/10.1371/journal.pone.0231101.s005

(PDF)

S2 Appendix. Minimal data set.

Values used to build graphs.

https://doi.org/10.1371/journal.pone.0231101.s006

(PDF)

References

  1. 1. Hamada S, Slade HD. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev. 1980; 44: 331–384. pmid:6446023
  2. 2. Bridge PD, Sneath PH. Streptococcus gallinarum sp. nov. and Streptococcus oralis sp. nov. Int J Syst Bacteriol. 1982; 32: 410–415.
  3. 3. Coykendall AL. Classification and identification of the viridans streptococci. Clin Microbiol Rev. 1989; 2: 315–328. pmid:2670193
  4. 4. Kolenbrander PE, London J. Adhere today, here tomorrow: oral bacterial adherence. J Bacteriol. 1993; 175: 3247–3252. pmid:8501028
  5. 5. Nobbs AH, Lamont RJ, Jenkinson HF. Streptococcus adherence and colonization. Microbiol Mol Biol Rev. 2009; 73: 407–450. pmid:19721085
  6. 6. Douglas CW, Heath J, Hampton KK, Preston FE. Identity of viridans streptococci isolated from cases of infective endocarditis. J Med Microbiol. 1993; 39: 179–182. pmid:8366515
  7. 7. Dyson C, Barnes RA, Harrison GA. Infective endocarditis: an epidemiological review of 128 episodes. J. Infect. 1999; 38: 87–93. pmid:10342647
  8. 8. Mitchell J. Streptococcus mitis: walking the line between commensalism and pathogenesis. Mol Oral Microbiol. 2011; 26: 89–98. pmid:21375700
  9. 9. Public Health England. Pyogenic and non-pyogenic streptococcal bacteraemia in England, Wales and Northern Ireland. Health Protection Reports [serial online]. 2013; 8: No.44. (doi is not appeared)
  10. 10. Kreth J, Zhang Y, Herzberg MC. Antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans. J Bacteriol. 2008; 190: 4632–4640. pmid:18441055
  11. 11. Chen L, Ge X, Dou Y, Wang X, Patel JR, Xu P. Identification of hydrogen peroxide production-related genes in Streptococcus sanguinis and their functional relationship with pyruvate oxidase. Microbiol, 2011; 157: 13–20.
  12. 12. Zhu L, Kreth J. The role of hydrogen peroxide in environmental adaptation of oral microbial communities. Oxid. Med. Cell. Longev. 2012; Article ID: 717843.
  13. 13. Redanz S, Cheng X, Giacaman RA, Pfeifer CS, Merritt J, Kreth J. Live and let die: hydrogen peroxide production by the commensal flora and its role in maintaining a symbiotic microbiome. Mol Oral Microbiol. 2018; 33: 337–352. pmid:29897662
  14. 14. Okahashi N, Nakata M, Sumitomo T, Terao Y, Kawabata S. Hydrogen peroxide produced by oral streptococci induces macrophage cell death. PLOS ONE. 2013; 8: e62563. pmid:23658745
  15. 15. Stone KD, Prussin C, Metcalfe DD. IgE, mast cells, basophils, and eosinophils. J Allergy Clin Immunol. 2010; 125: S73–S70. pmid:20176269
  16. 16. Galli SJ, Tsai M. IgE and mast cells in allergic disease. Nat Med. 2012; 18: 693–704. pmid:22561833
  17. 17. Eifan AO, Durham SR. Pathogenesis of rhinitis. Clin Exp Allergy. 2016; 46: 1139–1151. pmid:27434218
  18. 18. Krystel-Whittemore M, Dileepan KN, Wood JG. Mast cell: a multi- functional master cell. Front Immunol. 2016; 6: 620.
  19. 19. Walsh LJ. Mast cells and oral inflammation. Crit Rev Oral Biol Med. 2003; 14: 188–198. pmid:12799322
  20. 20. Gaje PN, Amalia Ceausu R, Jitariu A, Stratul SI, Rusu LC, Popovici RA, et al. Mast cells: key players in the shadow in oral inflammation and in squamous cell carcinoma of the oral cavity. Biomed Res Int. 2016; 2016: ID9235080.
  21. 21. Steiner M, Huber S, Harrer A, Himly M. The evolution of human basophil biology from neglect towards understanding of their immune functions. Biomed Res Int. 2016; 2016: 8232830. pmid:28078302
  22. 22. Calderón MA, Simons FE, Malling HJ, Lockey RF, Moingeon P, Demoly P. Sublingual allergen immunotherapy: mode of action and its relationship with the safety profile. Allergy. 2012; 67: 302–311. pmid:22150126
  23. 23. Barbuti G, Moschioni M, Censini S, Covacci A, Montecucco C, Montemurro P. Streptococcus pneumoniae induces mast cell degranulation. Int J Med Microbiol. 2006; 296: 325–329. pmid:16530009
  24. 24. Fritscher J, Amberger D, Dyckhoff S, Bewersdorf JP, Masouris I, Voelk S, et al. Mast cells are activated by Streptococcus pneumoniae in vitro but dispensable for the host defense against pneumococcal central nerve system infection in vivo. Front Immunol. 2018; 9: 550.
  25. 25. Watanabe Y, Todome Y, Ohkuni H, Sakurada S, Ishikawa T, Yutsudo T, et al. Cysteine protease activity and histamine release from the human mast cell line HMC-1 stimulated by recombinant streptococcal pyrogenic exotoxin B/streptococcal cysteine protease. Infect Immun. 2002; 70: 3944–3947. pmid:12065540
  26. 26. Gendrin C, Vornhagen J, Ngo L, Whidbey C, Boldenow E, Santana-Ufret V, et al. Mast cell degranulation by a hemolytic lipid toxin decreases GBS colonization and infection. Sci Adv. 2005; 1: e1400225.
  27. 27. Okahashi N, Okinaga T, Sakurai A, Terao Y, Nakata M, Nakashima K, et al. Streptococcus sanguinis induces foam cell formation and cell death of macrophages in association with production of reactive oxygen species. FEMS Microbiol Lett. 2011; 323: 164–170. pmid:22092716
  28. 28. Okahashi N, Sumitomo T, Nakata M, Sakurai A, Kuwata H, Kawabata S. Hydrogen peroxide contributes to the epithelial cell death induced by the oral mitis group of streptococci. PLOS ONE. 2014; 9: e88136. pmid:24498253
  29. 29. Sumioka R, Nakata M, Okahashi N, Li Y, Wada S, Yamaguchi M, et al. Streptococcus sanguinis induces neutrophil cell death by production of hydrogen peroxide. PLOS ONE. 2017; 12: e0172223. pmid:28222125
  30. 30. Matsushima H, Kumagai Y, Vandenbon A, Kataoka H, Kadena M, Fukamachi H, et al. Microarray analysis of macrophage response to infection with Streptococcus oralis reveals the immunosuppressive effect of hydrogen peroxide. Biochem Biophys Res Commun. 2017; 485: 461–467. pmid:28202416
  31. 31. Kulczycki A Jr, Isersky C, Metzger H. The interaction of IgE with rat basophilic leukemia cells. I. Evidence for specific binding of IgE. J Exp Med. 1974; 139: 600–616. pmid:4812630
  32. 32. Passante E, Ehrhardt C, Sheridan H, Frankish N. RBL-2H3 cells are an imprecise model for mast cell mediator release. Inflamm Res. 2009; 58: 611–618. pmid:19301096
  33. 33. Passante E, Frankish N. The RBL-2H3 cell line: its provenance and suitability as a model for the mast cell. Inflamm Res. 2009; 58: 737–745. pmid:19669619
  34. 34. Puri N, Kruhlak MJ, Whiteheart SW, Roche PA. Mast cell degranulation requires N-ethylmaleimide-sensitive factor-mediated SNARE disassembly. J Immunol. 2003; 171: 5345–5352. pmid:14607937
  35. 35. Kee JY, Inujima A, Andoh T, Tanaka K, Li F, Kuraishi Y, et al. Inhibitory effect of Moutan Cortex aqueous fraction on mast cell-mediated allergic inflammation. J Nat Med. 2015; 69: 209–217. pmid:25516374
  36. 36. Strobel S, Miller HRP, Ferguson A. Human intestinal mucosal mast cells: evaluation of fixation and staining techniques. J Clin Pathol. 1981; 34: 851–858. pmid:6168659
  37. 37. Falcieri E, Martelli AM, Bareggi R, Cataldi A, Cocco L. The protein kinase inhibitor staurosporine induces morphological changes typical of apoptosis in MOLT-4 cells without concomitant DNA fragmentation. Biochem Biophys Res Commun. 1993; 193: 19–25. pmid:8503906
  38. 38. Haenuki Y, Matsushita K, Futatsugi-Yumikura S, Ishii KJ, Kawagoe T, Imoto Y, et al. A critical role of IL-33 in experimental allergic rhinitis. J Allergy Clin Immunol. 2012; 130: 184–194. pmid:22460070
  39. 39. Kato Y, Akasaki S, Muto-Haenuki Y, Fujieda S, Matsushita K, Yoshimoto T. Nasal sensitization with ragweed pollen induced local-allergic-rhinitis-like symptoms in mice. PLOS ONE. 2014; 9: e103540. pmid:25119881
  40. 40. Hirose Y, Yamaguchi M, Goto K, Sumitomo T, Nakata M, Kawabata S. Competence-induced protein Ccs4 facilitates pneumococcal invasion into brain tissue and virulence in meningitis. Virulence. 2018; 9: 1576–1587. pmid:30251911
  41. 41. Okahashi N, Nakata M, Kuwata H, Kawabata S. Streptocccus oralis induces lysosomal impairment of macrophage via bacterial hydrogen peroxide. Infect. Immun. 2016; 84: 2042–2050. pmid:27113357
  42. 42. Spellerberg B, Cundell DR, Sandros J, Pearce BJ, Idanpaan-Heikkila I, Rosenow C, et al. Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae. Mol Microbiol. 1996; 19: 803–814. pmid:8820650
  43. 43. Braun JS, Sublett JE, Freyer D, Mitchell TJ, Cleveland JL, Tuomanen EI, et al. Pneumococcal pneumolysin and H2O2 mediate brain cell apoptosis during meningitis. J Clin Invest. 2002; 109: 19–27. pmid:11781347
  44. 44. Orihuela CJ, Gao G, Francis KP, Yu J, Tuomanen EI. Tissue-specific contribution of pneumococcal virulence factors to pathogenesis. J Infect Dis. 2004; 190: 1661–1669. pmid:15478073
  45. 45. Rai P, Parrish M, Tay IJ, Li N, Ackerman S, He F, et al. Streptococcus pneumoniae secretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells. Proc Natl Acad Sci. 2015; 112: E3421–3430. pmid:26080406
  46. 46. Loose M, Hudel M, Zimmer KP, Garcia E, Hammerschmidt S, Lucas R, et al. Pneumococcal hydrogen peroxide-induced stress signaling regulates inflammatory genes. J Infect Dis. 2015; 211: 306–316. pmid:25183769
  47. 47. Paton JC. The contribution of pneumolysin to the pathogenicity of Streptococcus pneumoniae. Trends Microbiol. 1996; 4: 103–106. pmid:8868088
  48. 48. Peden DB, Dailey L, DeGraff W, Mitchell JB, Lee JG, Kaliner MA, et al. Hydrogen peroxide effects on rat mast cell function. Am J Physiol. 1994; 267: L85–L93. pmid:8048546
  49. 49. Guerin-Marchand C, Sénéchal H, Pelletier C, Fohrer H, Olivier R, David B, et al. H2O2 impaired inflammatory mediator release from immunologically stimulated RBL-2H3 cells through a redox-sensitive, calcium-dependent mechanism. Inflamm Res. 2001; 50: 3341–3349.
  50. 50. Wan BY, Peh KH, Ho M, Assem ESK. Effects of nitric oxide and hydrogen peroxide on histamine release from RBL-2H3 cells. Biochem Pharm. 2001; 62: 1537–1544. pmid:11728390
  51. 51. Suzuki Y, Yoshimaru T, Inoue T, Niide O, Ra C. Role of oxidants in mast cell activation. Chem Immunol Allergy. 2005; 87: 32–42. pmid:16107761
  52. 52. Kaspar U, Kriegeskorte A, Schubert T, Peters G, Rudack C, Pieper DH, et al. The culturome of the human nose habitats individual bacterial fingerprint patterns. Enviroment Microbiol. 2016; 18: 2130–2142.
  53. 53. Bogaert D, van Belkum A, Sluijter M, Luijendijk A, de Groot R, Rümke HC, et al. Colonisation by Streptococcus pneumoniae and Staphylococcus aureus in health children. Lancet. 2004; 363: 1871–1872. pmid:15183627
  54. 54. Weiser JN, Ferreira DM, Paton JC. Streptococcus pneumoniae: transmission, colonization and invasion. Nat Rev Microbiol. 2018; 16: 355–367. pmid:29599457
  55. 55. Reber LL, Sibilano R, Mukai K, Galli SJ. Potential effector and immunoregulatory functions of mast cells in mucosal immunity. Mucosal Immunol. 2015; 8: 444–463. pmid:25669149
  56. 56. Metcalfe DD, Baram D, Mekori YA. Mast cells. Physiol Rev. 1997; 77: 1033–1079. pmid:9354811
  57. 57. Kawakami T, Galli SJ. Regulation of mast-cell and basophil function and survival by IgE. Nat. Rev. Immunol. 2002; 2: 773–786. pmid:12360215
  58. 58. Ivanov II, Honda K. Intestinal commensal microbes as immune modulators. Cell Host Microbe.2012; 12: 496–508. pmid:23084918
  59. 59. Hughes ER, Winter MG, Duerkop BA, Spiga L, Furtado de Carvalho T, Zhu W, et al, Microbial respiration and formate oxidation as metabolic signatures of inflammation-associated dysbiosis. Cell Host Microbe. 2017; 21: 208–219. pmid:28182951
  60. 60. Turk B, Turk V. Lysosomes as “suicide bags” in cell death: myth or reality? J Biol Chem. 2009; 284: 21783–21787. pmid:19473965
  61. 61. Mrschtik M, Ryan KM. Lysosomal proteins in cell death and autophagy. FEBS J. 2015; 282: 1858–1870. pmid:25735653
  62. 62. Zdolsek J, Zhang H, Roberg K, Brunk U. H2O2-mediated damage to lysosomal membranes of J-774 cells. Free Rad Res Commun. 1993; 18: 71–85.
  63. 63. Yu Z, Persson HL, Eaton JW, Brunk UT. Intralysosomal iron: a major determinant of oxidant-induced cell death. Free Rad Biol Med. 2003; 34: 1243–1252. pmid:12726912