Abstract

   Gut immune system homeostasis is crucial to overall host health. Immune
   disturbance at the gut level may lead to systemic and distant sites’
   immune dysfunction. Probiotics and prebiotics consumption have been
   shown to improve gut microbiota composition and function and enhance
   gut immunity. In the current study, the immunomodulatory and
   anti-inflammatory effects of viable and heat-inactivated forms of the
   novel probiotic bacterium Rouxiella badensis subsp. acadiensis (Canan
   SV-53), as well as the prebiotic protocatechuic acid (PCA) derived from
   the fermentation of blueberry juice by SV-53, were examined. To this
   end, female Balb/c mice received probiotic (viable or
   heat-inactivated), prebiotic, or a mixture of viable probiotic and
   prebiotic in drinking water for three weeks. To better decipher the
   immunomodulatory effects of biotics intake, gut microbiota, gut mucosal
   immunity, T helper-17 (Th17) cell-related cytokines, and epigenetic
   modulation of Th17 cells were studied. In mice receiving viable SV-53
   and PCA, a significant increase was noted in serum IgA levels and the
   number of IgA-producing B cells in the ileum. A significant reduction
   was observed in the concentrations of proinflammatory cytokines,
   including interleukin (IL)-17A, IL-6, and IL-23, and expression of two
   proinflammatory miRNAs, miR-223 and miR425, in treated groups. In
   addition, heat-inactivated SV-53 exerted immunomodulatory properties by
   elevating the IgA concentration in the serum and reducing IL-6 and
   IL-23 levels in the ileum. DNA methylation analysis revealed the role
   of heat-inactivated SV-53 in the epigenetic regulation of genes related
   to Th17 and IL-17 production and function, including Il6, Il17rc, Il9,
   Il11, Akt1, Ikbkg, Sgk1, Cblb, and Smad4. Taken together, these
   findings may reflect the potential role of the novel probiotic
   bacterium SV-53 and prebiotic PCA in improving gut immunity and
   homeostasis. Further studies are required to ascertain the beneficial
   effects of this novel bacterium in the inflammatory state.

   Keywords: gut immunity, gut microbiome, probiotic SV-53, prebiotic,
   Th17 cell, miRNA, DNA methylation

1. Introduction

   The largest immune system compartment in the body belongs to the gut
   [[42]1]. Paneth cells, Goblet cells, and gut-associated lymphoid tissue
   (GALT) contribute to the gut immune system function
   [[43]2,[44]3,[45]4]. Paneth cells produce antimicrobial peptides such
   as lysozyme and β-defensins, while Goblet cells produce mucus
   [[46]2,[47]3,[48]4]. Peyer’s patches, found in GALT, consist of B cell
   and T cell zones and are the main source of small intestine
   immunoglobulin A (IgA) plasmablasts [[49]3,[50]4]. The mucus layer,
   antimicrobial peptides, and IgA play crucial roles in gut barrier
   integrity and mucosal immunity [[51]5].

   Adaptive immune cells, including regulatory T cells (Treg) and T
   helper-17 (Th17), play a crucial role in maintaining gut immune system
   homeostasis. Signals from T cell receptors and cytokines direct T cells
   balance in the gut [[52]6]. Th17 cells are a distinct CD4^+ T-helper
   subtype characterized by interleukin (IL)-17 production that promotes
   inflammatory responses [[53]7]. Differentiation of naïve CD4^+ T cells
   into Th17 cells is regulated by the retinoic acid receptor-related
   orphan receptor-gamma-t (RORγt) transcriptional factor [[54]8].
   Transforming growth factor-β (TGF-β) and IL-6 are necessary for the
   early-stage differentiation of Th17 cells, while IL-23 plays a critical
   role in expanding Th17 cells and enhancing their pathogenic functions
   [[55]9,[56]10]. IL-10 inhibits Th17 cells development and immune
   responses [[57]11]. Over-production of Th17 cells is associated with
   autoimmunity and inflammatory diseases such as inflammatory bowel
   disease (IBD) [[58]6,[59]12].

   In addition to the role of cytokine milieu and transcription factors in
   the differentiation of naïve CD4^+ T cells [[60]13], the presence of
   Th17 in the small intestine is influenced by gut microbiota and
   microRNAs (miRNAs) profile [[61]14,[62]15,[63]16]. Gut miRNAs play
   pivotal roles in both innate and adaptive immunity, directing various
   physiological and immunological processes in the intestine. Specific
   miRNAs contribute to the proliferation of intestinal epithelial stem
   cells, epithelial regeneration, differentiation of Paneth and Goblet
   cells, and the balance between Th17 and Treg cells [[64]16].
   Additionally, epigenetic mechanisms, including DNA methylation and
   posttranslational histone modifications, contribute to T cell
   generation [[65]15].

   Probiotics and prebiotics have been shown to enhance gut immunity by
   beneficially affecting gut microbial communities, maintaining the gut
   epithelial barrier, inhibiting pathogens’ growth, and modulating innate
   and adaptive immune responses [[66]17,[67]18]. Probiotics have also
   been found to modulate the miRNAs expression involved in gut immunity
   [[68]19]. In addition, heat-inactivated probiotics, their fractions,
   and purified components have been demonstrated to confer health
   benefits by protecting against pathogens and enhancing intestinal
   barrier function [[69]20]. Probiotics and prebiotics may also enhance
   gut immunity by reducing proinflammatory cytokines such as IL-6 and
   IL-1β and increasing anti-inflammatory cytokines such as IL-10
   [[70]21]. Additionally, probiotics, prebiotics, and their metabolites
   have been shown to boost gut immunity by modulating epigenetic
   mechanisms, including DNA methylation and histone modification
   [[71]22,[72]23,[73]24].

   We have previously described the probiotic characteristics of a novel
   Gram-negative probiotic, Rouxiella badensis subsp. acadiensis (Canen
   SV-53), referred to as SV-53. This probiotic microorganism, which was
   isolated from the natural microflora of lowbush blueberry [[74]25],
   reinforces intestinal homeostasis by increasing the number of Paneth
   cells and production of the antimicrobial peptide α-defensin [[75]26].
   In the current study, the immunomodulatory properties of the live and
   heat-inactivated SV-53 and prebiotic protocatechuic acid (PCA) derived
   from fermented blueberry juice by SV-53, were studied in female Balb/c
   mice by analyzing the effects of biotics intake on gut microbiota, gut
   mucosal immunity, and selected cytokines and miRNAs involved in Th17
   cells differentiation and function. We also aimed to study whether
   SV-53 exerts potential immunomodulatory activities through modulating
   epigenetic mechanisms by analyzing its effect on the DNA methylation
   status of genes related to Th17 cell function.

2. Materials and Methods

2.1. Animals

   Eight-week-old female Balb/c mice (Charles River, Montreal, QC, Canada)
   were used in the current study. Three mice were housed together in
   plastic cages in a controlled atmosphere (temperature 22 ± 2 °C;
   humidity 55 ± 2%) with a 12 h light/dark cycle. During the study, all
   groups received a conventional balanced diet ad libitum. Mice were
   maintained and treated according to the guidelines of the Canadian
   Council on Animal Care. The protocol (HSe-3191) was approved by the
   Animal Care Committee of the University of Ottawa.

2.2. Probiotic and Prebiotic Solution Preparation

   In the current study, we used the research bank cultures of the
   bacterium prepared by the National Research Council of Canada. The
   bacteria cultures were maintained in a tryptic soy broth (TSB) culture
   medium (Difco Laboratories, Detroit, MI, USA) supplemented with 30%
   (v/v) glycerol at −80 °C.

   To prepare the probiotic solution, the bacteria were cultured in TSB
   for 17 h at room temperature. Then, the bacteria culture was
   centrifuged at 5000 rpm for 10 min. The bacterial pellet was washed
   three times in sterile PBS (Sigma, Saint Louis, MO, USA) and
   resuspended in 5 mL of sterile 10% (wt/vol) non-fat milk. Bacterial
   suspensions were diluted 1:30 in water and administered ad libitum to
   the mice at a final concentration of 5 × 10^7 CFU/mL. To prepare
   heat-inactivated bacteria, the same procedure was applied, and 5 × 10^7
   CFU/mL bacterial preparation in the sterile water was heated at 70 °C
   for 30 min. The heat-inactivated preparations were kept at −80 °C to
   feed the mice. An average of 2 mL of probiotic solution was consumed
   daily by each mouse; therefore, the mice approximately received a daily
   dose of 1 × 10^8 CFU of the live and heat-inactivated bacterium.

   To prepare the prebiotic solution, 100 mg/kg BW of PCA
   (3,4-dihydroxybenzoic acid) was dissolved in sterile water. Then, the
   pH of the prebiotic solution was adjusted to 7.4 using a pH meter. The
   fresh mixture was prepared twice a week and kept at 4 °C.

2.3. Study Design

2.3.1. Probiotic-Prebiotic Experiment

   Mice (n = 24) were categorized into four groups: 1—control group;
   receiving sterile water, 2—probiotic group; receiving probiotic SV-53
   (5 × 10^7 CFU/mL) in sterile drinking water, 3—prebiotic group;
   receiving 100 mg/kg prebiotic PCA in sterile drinking water,
   4—probiotic + prebiotic group; receiving a mixture of SV-53 and PCA in
   sterile drinking water. The duration of the nutritional intervention
   was three weeks. Afterward, mice were sacrificed, and the required
   samples, including blood, feces, and ileum tissues, were collected and
   stored at −80 °C to conduct corresponding experiments.

2.3.2. Heat-Inactivated Probiotic Experiment

   Mice (n = 18) were divided into three groups: 1—control; receiving
   sterile drinking water, 2—probiotic group; receiving live SV-53 (5 ×
   10^7 CFU/mL) in sterile drinking water, and 3—heat-inactivated
   probiotic group; receiving heat-inactivated SV-53 (5 × 10^7 CFU/mL) in
   sterile drinking water. At the end of three weeks of nutritional
   intervention, mice were sacrificed, and the required samples were
   collected and kept at −80 °C until further experiments were performed.

2.4. Histological Sections Preparation

   The ileum tissues of mice were removed, washed with ice-cold PBS, and
   small sections of the ileum were collected and fixed in a 4%
   paraformaldehyde solution for 48 h. Subsequently, fixed tissues were
   dehydrated in increasing alcohol concentrations, cleared in xylene, and
   embedded in paraffin using conventional methods. Histological sections
   of 4 µm were prepared from paraffin blocks using a rotational microtome
   (Leica RM2255 Automated Microtome). The processing, embedding, and
   preparation of histological sections were performed by the University
   of Ottawa Histology Core Facility.

2.5. Identification of IgA, IgG, IL-17A, IL-6, IL-23, and IL-10 Producing
Cell Populations by Immunofluorescence

   The numbers of IgA^+, IgG^+, and IL-17A^+ cells on histological
   sections from the ileum region were determined via direct
   immunofluorescence. The immunofluorescence tests were performed using
   FITC-conjugated goat (α-chain specific) polyclonal anti-mouse IgA
   (Sigma-Aldrich, St. Louis, MO, USA), FITC-conjugated goat (γ-chain
   specific) polyclonal anti-mouse IgG (Sigma-Aldrich, St. Louis, MO,
   USA), and anti-Mo/Rat IL-17A (ebio17B7), (eBioscience, Thermo Fisher
   Scientific, Burlington, ON, Canada). The histological sections were
   deparaffinized in xylene and rehydrated in a graded series of ethanol
   from 95% to 40%. The deparaffinized histological samples were stained
   with appropriate antibody dilution in 1X PBS (1:100 for IgA, 1:50 for
   IgG, and IL-17A) for 1 h at 37 °C. Indirect immunofluorescence was used
   to determine the number of IL-6 and IL-10-producing cells using
   polyclonal anti-murine IL-10 (PeproTech, Rocky Hill, NJ, USA) and
   polyclonal anti-murine IL-6 (PeproTech, Rocky Hill, NJ, USA)
   antibodies, respectively. The deparaffinized histological slides were
   incubated with appropriate primary antibody dilution in 1X PBS (1:50)
   overnight at 4 °C and FITC-conjugated affinipure goat anti-rabbit IgG
   (H + L) secondary antibody (1:50 in 1X PBS) for 1 h at 37 °C in the
   dark. The slides were washed three times in PBS, mounted using
   Fluoromount (Sigma-Aldrich, St. Louis, MO, USA), and examined using a
   fluorescent light microscope (Evos FL Auto 2, Thermo Fisher Scientific,
   Bothell, WA, USA). The results were expressed as the number of positive
   cells (fluorescent cells) per 10 fields at 40× magnification.

2.6. Determination of IgA, IgG, IL-17A, IL-6, IL-23, and IL-10 Concentrations
by ELISA

   Blood samples were collected and centrifuged at 10,000× g for 2 min to
   separate serum. Serum samples were kept at −80 °C. Small pieces of
   ileum were snap-frozen and kept at −80 °C until protein extraction. To
   examine the cytokine levels, small parts of the ileum tissues (20–25 g)
   were collected in microtubes containing lysis buffer (Pierce IP Lysis,
   Thermo Fisher Scientific) and protease/phosphatase inhibitors (Halt
   Protease and Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific)
   and homogenized using an electrical homogenizer (Bead mill 24, Fisher
   Scientific, USA). IgA and IgG levels were determined in serum using
   mouse IgA and IgG uncoated ELISA kits (Invitrogen, Vienna, Austria).
   The levels of IL-6, IL-10, IL-17A, and IL-23 were measured in ileum
   tissues using mouse-uncoated ELISA kits (Invitrogen, Vienna, Austria).
   All ELISA tests were performed according to the manufacturer’s
   instructions. Absorbance was read at 450 nm with a wavelength
   correction of 570 nm using a microplate reader (Bio-TEK Instruments,
   Winooski, VT, USA).

2.7. Determination of miRNA Expression by Real-Time Quantitative Reverse
Transcription PCR (RT-qPCR)

   Small pieces of ileum were placed in tubes containing RNAlater
   Stabilization Solution (Invitrogen, Carlsbad, CA, USA) for 24 h and
   then stored at −80 °C until RNA extraction. Total RNA from the samples
   was extracted using miRNeasy mini kit (Qiagen, Toronto, ON, Canada).
   Samples purity was verified with a NanoDrop 2000 (Thermo Scientific,
   Waltham, MA, USA). A reverse transcription reaction was performed to
   synthesize cDNA using the miRCURY LNA RT Kit (Qiagen, Toronto, ON,
   Canada). The expression of miR-223 and miR-425 was measured via RT-qPCR
   using hsa-miR-425-5p and hsa-miR-223-3p miRCURY LNA miRNA PCR assay
   primers (Qiagen, Toronto, ON, Canada) and miRCURY LNA SYBR Green PCR
   Kit (Qiagen, Toronto, ON, Canada) in a CFX 384 real-time PCR detection
   system (Bio-Rad, Laboratories, Hercules, CA, USA). miRNA expression was
   normalized to U6 as the reference gene using the U6 snRNA (hsa, mmu)
   miRCURY LNA miRNA PCR assay (Qiagen, Toronto, ON, Canada).

2.8. Gut Microbiome Analysis

   For microbiome analysis, cecum contents of mice were collected in
   sterile microtubes and snap-frozen in liquid nitrogen before storage at
   −80 °C. Shallow shotgun sequencing was used for microbiome analysis.
   Microbiome analysis was performed by Microbiome Insights Company,
   Vancouver, BC. DNA was extracted using the MagAttract PowerSoil DNA KF
   kit (Qiagen, Toronto, ON, Canada). The quality of extracted DNA was
   evaluated visually through gel electrophoresis and quantified using a
   Qubit 3.0 fluorometer (Thermo-Fischer, Waltham, MA, USA). Libraries
   were prepared using an Illumina Nextera library preparation kit with an
   in-house protocol (Illumina, San Diego, CA, USA).

2.9. Methylome-Wide Profiling and Data Analysis

   Approximately 15–20 g of the mice ileum samples was homogenized using
   an electrical homogenizer in tubes containing 500 µL cell lysis buffer
   and 1.5 µL proteinase K. The Gentra Puregene Tissue Kit (33 g) (Qiagen,
   Toronto, ON, Canada) was then employed to extract DNA from the tissues,
   according to the manufacturer’s instructions. The extracted DNA was
   quantified using Qubit 4 (Thermo Fisher Scientific, Waltham, MA, USA)
   and diluted with DNA rehydration solution to achieve a final
   concentration of 20 ng/µL, then stored at −20 °C. Methylome-wide
   profiling was conducted as previously described [[76]27]. Briefly, 500
   ng of extracted DNA was subjected to bisulfite conversion using the EZ
   DNA Methylation kit (Zymo Research, Irvine, CA, USA). Next, 250 ng
   bisulfite-modified DNA was analyzed using the Infinium Mouse
   Methylation BeadChip arrays, which allow for the simultaneous
   assessment of DNA methylation at more than 285,000 CpG sites (Illumina
   Inc., San Diego, CA, USA). Methylome-wide data were analyzed using the
   methylkey pipeline developed by the Epigenomics and Mechanisms Branch
   at the International Agency for Research on Cancer
   ([77]https://github.com/IARCbioinfo/methylkey (accessed on 21 January
   2023)). Briefly, raw data files were pre-processed, quality control was
   conducted, and normalization was performed via Noob normalization using
   the SeSAMe package [[78]28]. Intergroup comparisons were conducted
   using linear regression analysis as implemented in the limma R package
   [[79]29]. Regional analysis to identify differentially methylated
   regions was conducted using the DMRcate package [[80]30].

2.10. Statistical Analysis

   All statistical analyses, except for the microbiome analysis, were
   conducted using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA,
   USA). One-way analysis of variance (ANOVA) followed by Dunnett’s
   multiple comparisons test was employed to compare groups. Data were
   considered significantly different when the p-value < 0.05. All values
   are mean ± SEM, based on at least three independent tests. Microbiome
   analysis was performed using Qiime2, and for comparisons of
   differentially abundant taxa, negative binomial models (DESEq2 R
   package) were utilized. Alpha diversity was calculated from taxonomic
   profiles using Shannon’s diversity index. Beta diversity analysis was
   conducted using Bray–Curtis dissimilarities, and the results were
   visualized using nonmetric multidimensional scaling (NMDS). A
   corrected/adjusted p-value < 0.05 was considered statistically
   significant. For Methylation analysis, differentially methylated genes
   were defined with a false discovery rate (FDR)-adjusted p-value < 0.05
   and an absolute inter-group beta value difference of >0.05. Pathway
   visualization was performed using KEGG pathway enrichment analysis with
   Enrichr.

3. Results

3.1. Effect of Probiotic and Prebiotic Intake on Mucosal Immunity

   The effect of the nutritional intervention on mucosal immunity was
   assessed by measuring IgA and IgG levels in the serum, as well as the
   population of IgA and IgG-producing cells in the lamina propria of the
   ileum tissues. Feeding mice with SV-53 and SV-53 + PCA mixture
   significantly increased the serum IgA level (p < 0.05) ([81]Figure 1A).
   Furthermore, the population of IgA^+ cells exhibited a significant
   increase in the ileum tissues of treated groups compared to the
   untreated group (p < 0.05, p < 0.01, and p < 0.0001, respectively)
   ([82]Figure 1B). However, neither the serum IgG level nor the number of
   IgG+ cells in the ileum of mice changed following the feeding period
   ([83]Figure 1C,D). [84]Figure 1E illustrates immunofluorescence images
   of histological sections stained with FITC-conjugated anti-IgA
   antibody.

Figure 1.

   [85]Figure 1
   [86]Open in a new tab

   The effect of the probiotic and prebiotic intake on gut mucosal
   immunity. Female Balb/c mice were treated with the SV-53, PCA, or SV-53
   + PCA mixture administered in their drinking water for three weeks.
   Serum levels of IgA and IgG and the number of IgA^+ and IgG^+ B cells
   in the ileum tissues of mice were measured via ELISA and direct
   immunofluorescence, respectively; (A) the serum concentration of IgA,
   (B) the number of IgA^+ B cells, (C) the serum concentration of IgG,
   and (D) the number of IgG^+ B cells. One-way ANOVA followed by
   Dunnett’s multiple comparisons were used to compare groups. All values
   are mean ± SEM. N = 6, * p < 0.05, ** p < 0.01 and **** p < 0.0001 vs.
   control. (E) Immunofluorescence images of histological sections of the
   ileum stained with the appropriate dilution of anti-IgA antibody
   (1:100) and imaged using a fluorescent light microscope at 40×
   magnification.

3.2. Effect of Probiotic and Prebiotic Intake on the Expression of Selected
Cytokines and miRNAs

   Oral administration of SV-53 and PCA for three weeks led to a
   significant reduction in IL-17A (p < 0.01, p < 0.05, and p < 0.01,
   respectively) ([87]Figure 2A), IL-6 (p < 0.01, p < 0.05, and p < 0.01,
   respectively) ([88]Figure 2B), and IL-23 (p < 0.01, p < 0.05, and p <
   0.001, respectively) ([89]Figure 2C) concentrations in the ileum
   tissues of treated mice compared to the control counterparts. The level
   of IL-10 increased in all treatment groups; however, statistical
   significance was reached only in the prebiotic group (p < 0.05)
   ([90]Figure 2D).

Figure 2.

   [91]Figure 2
   [92]Open in a new tab

   The effect of the probiotic and prebiotic intake on selected cytokines
   and miRNAs in the ileum tissues of mice. Female Balb/c mice were
   treated with the SV-53, PCA, or SV-53 + PCA mixture in drinking water
   for three weeks. The concentrations of (A) IL-17A, (B) IL-6, (C) IL-23,
   and (D) IL-10 in the ileum tissues of mice were measured via ELISA. The
   frequencies of (E) IL-17A^+, (F) IL-6^+, and (G) IL-10^+ cells were
   determined via immunofluorescence. The expression of (H) miR-223 and
   (I) miR-425 was measured via RT-qPCR. One-way ANOVA followed by
   Dunnett’s multiple comparisons was used to compare groups. All values
   are mean ± SEM. N = 6, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs.
   control.

   Additionally, the number of IL-17A, IL-6, and IL-10-producing cells in
   the lamina propria was measured through immunofluorescence. In
   accordance with ELISA results, the quantity of IL-17A-producing cells
   was significantly lower in all treatment groups than in the control (p
   < 0.01, p < 0.05, and p < 0.01, respectively) ([93]Figure 2E). IL-6
   producing cells frequency was reduced in mice fed SV-53 + PCA mixture
   compared to the control (p < 0.05) ([94]Figure 2F), while the abundance
   of IL-10-producing cells increased in SV-53 and PCA-fed mice (p < 0.01
   and p < 0.05, respectively) ([95]Figure 2G).

   Furthermore, the impact of SV-53 and PCA intake on the expression of
   two pro-inflammatory miRNAs, miR-223 and miR-425, was analyzed. Feeding
   mice with SV-53, PCA, and their mixture significantly decreased the
   expression of miR-223 (p < 0.05) and miR-425 (p < 0.01, p < 0.001, and
   p < 0.001, respectively) in ileum tissues of mice compared to the
   control group ([96]Figure 2H,I).

3.3. Effect of the Probiotic and Prebiotic Intake on the Gut Microbiome

   Alpha diversity, calculated from taxonomic profiles using Shannon’s
   diversity index, did not show significant differences across groups
   ([97]Figure 3A). Beta diversity was assayed using Bray–Curtis
   dissimilarities and samples were visualized using nonmetric
   multidimensional scaling (NMDS). Samples were very similar in general
   and no significant difference was observed ([98]Figure 3B). [99]Figure
   3C displays the ten most abundant phyla in all groups. Microbiome
   analysis revealed the intestinal colonization of SV-53 in the mice that
   received SV-53 and SV-53 + PCA (p < 0.0001) ([100]Figure 3D) and a
   significant decrease in the abundance of Escherichia coli (E. coli) in
   the SV-53 and SV-53 + PCA groups (p < 0.0001) ([101]Figure 3E).

Figure 3.

   [102]Figure 3
   [103]Open in a new tab

   The effect of the probiotic and prebiotic intake on the gut microbiome.
   Female Balb/c mice were treated with the SV-53, PCA, or SV-53 + PCA
   mixture in drinking water for three weeks. Then, cecum contents of mice
   were used to analyze the gut microbiome via shallow shotgun sequencing;
   (A) alpha diversity, (B) beta diversity, (C) bacterial composition at
   the phylum level (ten most abundant phyla), and (D,E) differentially
   abundant species. n = 5 in the control group and n = 6 in the treatment
   groups.

3.4. Effect of Heat-Inactivated SV-53 Intake on Mucosal Immunity

   The impact of heat-inactivated SV-53 on gut mucosal immunity was
   investigated in a separate experiment. Three weeks of treatment with
   heat-inactivated bacteria led to a significant increase in serum IgA
   levels (p < 0.05) with no significant effect on the IgA^+ cells
   population in the lamina propria ([104]Figure 4A,B, respectively).
   Heat-inactivated bacteria also had no significant effect on IgG levels
   and the population of IgG^+ cells in lamina propria ([105]Figure 4C,D,
   respectively).

Figure 4.

   [106]Figure 4
   [107]Open in a new tab

   The effect of the heat-inactivated probiotic intake on gut mucosal
   immunity. Female Balb/c mice were treated with live SV-53 or
   heat-inactivated SV-53 (HI-SV-53) in drinking water for three weeks.
   Then, the serum levels of IgA and IgG, and the number of IgA^+ and
   IgG^+ B cells in the ileum tissues of mice were measured by ELISA and
   immunofluorescence, respectively; (A) the concentrations of IgA, (B)
   the number of IgA^+ B cells, (C) the concentrations of IgG, and (D) the
   number of IgG^+ B cells. One-way ANOVA followed by Dunnett’s multiple
   comparisons were used to compare groups. All values are mean ± SEM. N =
   6, * p < 0.05 and *** p < 0.001 vs. control.

3.5. Effect of Heat-Inactivated SV-53 Intake on the Expression of Selected
Cytokines and miRNAs

   IL-17A levels in the ileum tissues of mice were insignificantly lower
   in the heat-inactivated SV-53 group than those in the control group
   ([108]Figure 5A). However, IL-6 and IL-23 concentrations significantly
   decreased in the ileum tissue of mice following treatment with
   heat-inactivated SV-53 (p < 0.05 and p < 0.01, respectively)
   ([109]Figure 5B,C). The heat-inactivated SV-53 treatment had no
   significant effect on the IL-10 level in the ileum of mice ([110]Figure
   5D). Administration of heat-inactivated SV-53 significantly reduced the
   numbers of IL-17A^+ and IL-6^+ cells compared to the control (p < 0.05)
   ([111]Figure 5E,F, respectively), while it did not change the
   IL-10-producing cells’ abundance ([112]Figure 5G). Additionally, the
   administration of heat-inactivated bacteria did not alter the
   expression of miR-223 and miR-425 in the ileum tissues of mice compared
   to the untreated group ([113]Figure 5H,I).

Figure 5.

   [114]Figure 5
   [115]Open in a new tab

   The effect of the heat-inactivated probiotic on selected cytokines and
   miRNAs expression in the ileum tissues of mice. Female Balb/c mice were
   treated with live SV-53 or heat-inactivated SV-53 (HI-SV-53) in
   drinking water for three weeks. The concentrations of (A) IL-17A, (B)
   IL-6, (C) IL-23, and (D) IL-10 in the ileum tissues of mice were
   measured via ELISA. The frequencies of (E) IL-17A^+, (F) IL-6^+, and
   (G) IL-10^+ cells were determined via immunofluorescence. The
   expression of (H) miR-223 and (I) miR-425 were measured via RT-qPCR.
   One-way ANOVA followed by Dunnett’s multiple comparisons was used to
   compare groups. All values are mean ± SEM. N = 6, * p < 0.05, ** p <
   0.01 and *** p < 0.001 vs. control.

3.6. Effect of Heat-Inactivated SV-53 on DNA Methylation Status

   Finally, the impact of heat-inactivated SV-53 on the methylation status
   of genes in the ileum samples of mice was analyzed. The methylation
   status of regions around the transcriptional start sites (TSS) plays a
   role in regulating gene expression [[116]31,[117]32], where
   hypomethylation and hypermethylation correlate with transcriptional
   activation and suppression, respectively [[118]33].

   Absolute group mean difference in beta values of >0.05 and FDR adjusted
   p-value < 0.05 were applied to identify differentially methylated
   probes (DMPs), which were used as a basis for regional analysis to find
   statistically significant differentially methylated regions (DMRs). We
   found significant DMRs related to different signaling pathways such as
   MAPK, PI3K-Akt, JAK-STAT, forkhead box O (FoxO), Th17 differentiation,
   and IL-17 signaling pathways. [119]Figure 6A illustrates the top
   enriched pathways for genes associated with the identified DMRs.
   Notably, significant alterations in the methylation status of some
   genes regulating Th17 differentiation and function were observed in
   mice receiving heat-inactivated SV-53 compared to the control
   ([120]Figure 6B). Significant hypermethylation of CpGs was identified
   within the promoters of Il6, IL-17 receptor c (Il17rc), and Il9, and
   the 1 to 5 kb region of Il11. In addition, the 1 to 5 kb region of Akt1
   and the promoters of inhibitor of nuclear factor kappa B kinase
   regulatory subunit gamma (Ikbkg) and serum glucocorticoid kinase 1 gene
   (Sgk1) were significantly hypermethylated. Furthermore, the 5′ UTR
   region of casitas-B-lineage lymphoma protein-b (Cblb) and the 1 to 5 kb
   region of SMAD family member 4 (Smad4) were found to be hypomethylated
   in the heat-inactivated SV-53 group compared to the control group. No
   significant DMRs were found in mice receiving live SV-53 and PCA
   compared to untreated mice.

Figure 6.

   [121]Figure 6
   [122]Open in a new tab

   The effect of the heat-inactivated probiotic on DNA methylation. Female
   Balb/c mice were treated with live SV-53 or heat-inactivated SV-53
   (HI-SV-53) administered via drinking water for three weeks. (A)
   Pathways enrichment analysis visualized by Enrichr, (B) boxplots of
   differentially methylated genes related to Th17 cells differentiation
   and function. * p < 0.05 and ** p < 0.01 vs. control.

4. Discussion

   Probiotics, prebiotics, and symbiotic products have been shown to
   promote a balanced gut microbiota, reduce inflammation, and enhance
   immune system function—not only gut immunity but also systemic and
   distant tissue immunity through the common mucosal immune system
   [[123]1,[124]34,[125]35,[126]36,[127]37]. In this study, the
   immunomodulatory and anti-inflammatory properties of the novel
   probiotic SV-53 and prebiotic PCA were explored. Certain probiotic
   bacteria and prebiotics, including small polyphenol oligomers, act as
   antagonist ligands for Toll-like receptors (TLRs), effectively
   inhibiting associated inflammatory pathways [[128]38,[129]39]. SV-53 is
   a Gram-negative bacteria with probiotic characteristics [[130]26]. The
   subtle distinction between bacterial lipopolysaccharides (LPS) derived
   from pathogenic Gram-negative bacteria and Gram-negative commensal
   bacteria in gut microbiota is responsible for the immunoinhibitory
   activity of Gram-negative commensals [[131]40]. On the other hand, PCA
   is a metabolite product resulting from the fermentation of blueberry
   juice by SV-53 and deriving from complex polyphenols and quercetin
   [[132]38], which is a TLR4 antagonist [[133]41].

   The results of the current study revealed that SV-53 and PCA
   administration could increase the population of IgA-producing B cells
   in the ileum, and SV-53 intake increased serum IgA concentration
   without increasing IgG levels. Within the intestinal environment,
   IgA-positive (IgA^+) cells and secretory IgA (sIgA) play a vital role
   in maintaining intestinal mucosal immunity and homeostasis [[134]42].
   sIgA is the most abundant antibody class in the mucosal immune system
   and gut lumen [[135]43]. It enhances gut mucosal immunity and
   homeostasis through various mechanisms, including quenching microbial
   components, neutralizing bacterial toxins, influencing gut microbial
   communities, enhancing antigens transport to immune cells in GALT, and
   downregulating proinflammatory responses [[136]44]. On the other hand,
   IgG antibodies are potent effector molecules that activate innate
   immune cells and induce inflammation [[137]45,[138]46]. An increased
   IgG level is associated with intestinal inflammation and persistent
   immunopathology in the intestinal mucosa [[139]47]. In addition, an
   increase in local commensal-specific IgG during intestinal inflammatory
   diseases, such as colitis, has been reported by Castro-Dopico et al.
   (2019), and this increase was associated with the magnitude of
   intestinal inflammation [[140]48]. They also found that IgG immune
   complexes-stimulated colonic macrophages demonstrated a Th17-polarizing
   phenotype [[141]48]. No changes in IgG levels were observed in response
   to probiotic intake, reflecting the effectiveness of the treatment in
   terms of improving gut mucosal immunity by producing sIgA without
   increasing IgG levels and inducing inflammation.

   In addition, probiotic and prebiotic intake significantly decreased the
   level of IL-17A, the main proinflammatory cytokine secreted by Th17
   cells [[142]7], and levels of IL-6 and IL-23, major cytokines inducing
   pathogenic Th17 cells production and function, in the ileum tissues of
   mice [[143]9,[144]10]. IL-17A is involved in the pathogenicity of
   inflammatory disorders [[145]10]. For instance, the involvement of
   IL-17A/IL-23 in the pathogenesis of IBD has been shown in both human
   and animal studies [[146]49]. We have previously shown that the
   fermentation of blueberry juice with SV-53 significantly increases the
   quantity of polyphenols naturally present in the juice [[147]50]. This
   novel product was found to inhibit IL-6/STAT3 signaling [[148]51].
   IL-6/STAT3/RORγt pathway induces Th17 cells differentiation [[149]52],
   and IL-23/IL-23 receptor signaling phosphorylates the STAT3 and
   increases the expression of inflammatory cytokines such as IL-17A,
   leading to intestinal inflammation [[150]53]. Moreover, increased IL-6
   negatively affects gut mucosal immunity and leads to gut barrier
   dysfunction by damaging tight junctions [[151]54,[152]55]. Our results
   may provide some evidence regarding the potential role of SV-53 and PCA
   in modulating the IL6/STAT3 pathway. However, further studies are
   required to explore the role of SV-53 and PCA in regulating the STAT3
   pathway.

   Furthermore, we found an increase in the IL-10 concentration in the PCA
   group and an increase in the number of IL-10-producing cells in the
   SV-53 and PCA groups. IL-10 is an immunosuppressive cytokine secreted
   by various immune cells, including dendritic cells, macrophages, and
   Tregs. IL-10-producing CD4^+ CD25^+ Treg cells play a key role in
   controlling intestinal inflammation and self-tolerance [[153]56]. IL-10
   contributes to B cells differentiation and the production of IgA by B
   cells, thereby enhancing mucosal humoral immunity [[154]57]. In
   addition, in a study using a chronic colitis mice model, IL-10
   suppressed the inflammasome/IL-1β pathway, reduced the pathogenicity of
   Th17 cells, and suppressed IL-17 production, leading to the mitigation
   of intestinal inflammation [[155]58]. Lactic acid bacteria have been
   shown to prevent intestinal inflammation and autoimmunity by increasing
   IL-10 and CD4^+ CD25^+ Treg cells [[156]59,[157]60].

   Moreover, studies have shown that prebiotics have the ability to
   modulate IgA, IgG, proinflammatory, and anti-inflammatory cytokines
   [[158]61]. For example, prebiotic intake increased sIgA and IL-10
   levels in the intestines of rats [[159]62] and inhibited LPS-induced
   IL-17 release in mouse splenocytes [[160]63]. Additionally, polyphenols
   inhibit inflammation by blocking TLR4 and suppressing the production of
   inflammatory mediators such as IL-1β, IL-6, and TNFα [[161]61].

   Gut miRNAs link the gut immune system and the microbial community by
   regulating signaling pathways critical for maintaining gut
   microenvironment homeostasis [[162]64]. In the current study, the
   effect of nutritional intervention with probiotic SV-53 and prebiotic
   PCA intake on miR-223 and miR-425 expression was explored, and a
   significant decrease in the relative expression of both miRNAs was
   observed in the treatment groups. A marked increase in the expression
   of miR-223 and miR-425 has been reported in intestinal inflammatory
   conditions such as IBD [[163]65,[164]66]. miR-223 may promote the
   secretion of cytokines by dendritic cells and therefore regulate Th17
   and Tregs differentiation from naïve T cells [[165]64]. miR-233 is
   downstream of the IL-23 cascade [[166]67] and has been shown to promote
   pathological Th17 cells differentiation during autoimmunity [[167]68].
   miR-223 also increases intestinal barrier permeability by targeting
   thigh junction proteins by mediating the IL23/Th17 pathway [[168]67].
   Impaired barrier function is a major factor contributing to intestinal
   inflammation [[169]67]. miR-425 is involved in chronic–degenerative
   inflammations such as autoimmune diseases, age-related disorders, and
   cancer progression [[170]69]. This miRNA promotes Th17 cells
   differentiation and pathogenicity. In a study conducted on colitic
   mice, overexpression of miR-425 was found to mediate Th17 cell
   production and IL-17A secretion, while inhibiting miR-425 alleviated
   intestinal mucosal inflammation and markedly decreased IL-17A levels
   [[171]65]. Therefore, these results suggest that SV-53 and PCA may
   enhance gut immunity and prevent inflammation by regulating gut miRNAs
   as well.

   Similar to these results, several studies have reported the beneficial
   effects of probiotics in mitigating gut inflammation through the
   modulation of miRNA expression. For instance, the oral administration
   of two probiotics, Lactobacillus fermentum and Saccharomyces boulardii,
   significantly regulated the expressions of miR-223 and improved gut
   barrier function, restored dysbiosis, and ameliorated disease severity
   in a mouse model of dextran sodium sulfate (DSS)-induced colitis
   [[172]70,[173]71]. In another study, feeding mice with probiotic E.
   coli Nissle 1917, a Gram-negative probiotic bacterium, prevented the
   DSS-induced colonic inflammation by enhancing the expression of various
   cytokines and proteins responsible for maintaining epithelial integrity
   and decreasing several miRNAs involved in the inflammatory response,
   including miR-223 [[174]66].

   Full genome analysis of SV-53 has revealed a cluster of genes coding
   for bacteriocins against pathogenic Gram-negative bacteria [[175]72].
   We have previously shown the antibacterial effect of SV-53 against
   Staphylococcus aureus and Salmonella enterica serovar Typhimurium
   [[176]26]. In this study, the microbiome analysis signified a decrease
   in E. coli population in mice fed SV-53 and the mixture of SV-53 and
   PCA. An increase in pathogenic E. coli has been observed in IBD and
   following antibiotic-induced dysbiosis [[177]73,[178]74]. Furthermore,
   studies have observed that the Western diet can alter gut microbiota
   composition, increase gut permeability, induce overgrowth of E. coli,
   promote invasive E. coli colonization in the gut mucosa, and
   subsequently lead to inflammation and immune dysfunction
   [[179]75,[180]76]. Moreover, enterotoxigenic E. coli exposure has been
   shown to increase RORγt expression and IL-17 secretion in the
   intestines of mice [[181]77]. Several probiotics, such as Lactobacillus
   plantarum and E. coli Nissle 1917, have been reported to alleviate
   pathogenic E. coli-induced intestinal barrier dysfunction by modulating
   the expression of tight junction proteins [[182]78].

   Furthermore, we demonstrated the immunomodulatory and anti-inflammatory
   properties of heat-inactivated SV-53 by increasing IgA concentration in
   the serum and reducing IL-6 and IL-23 concentrations and the number of
   IL-17A and IL-6 producing cells in the ileum tissues of mice. However,
   our results indicated that viable SV-53 exhibited more robust
   immunomodulatory properties compared to the non-viable bacterium. Many
   of the ascribed benefits of live probiotics are facilitated by
   interactions of these organisms with the host’s gut epithelium and
   immune system. They can interact with the host directly through
   cell-to-cell interaction or indirectly through the production of
   metabolites and the release of various components [[183]79].
   Heat-inactive probiotics are also metabolically active and can interact
   with the host, resulting in health benefits [[184]79]. In fact,
   heat-killed probiotics retain functionality due to the presence of cell
   wall components involved in interactions with the host, such as
   lipoteichoic acids and peptidoglycan [[185]79].

   In an experimental colitis model, the administration of a mixture of
   heat-inactivated probiotics downregulated IL-6, IL-23, STAT3, and
   p-STAT3 expression in colonic tissues of colitic rats [[186]80]. In
   another study, heat-inactivated E. coli Nissle 1917 induced the
   antimicrobial peptide β-defensin in intestinal epithelial cells
   [[187]81]. We have previously shown the immunomodulatory and
   anti-inflammatory activity of heat-inactivated lactic acid bacteria
   using primary cultures of intestinal epithelial cells in mice fed
   Lactobacillus casei CRL 431 or Lactobacillus helveticus R389 [[188]82].
   Similar to this study, Pyclik et al. demonstrated the significance of
   the viability status of Bifidobacterium strains for its
   immunomodulatory properties, with heat inactivation being shown to
   alter these properties [[189]83]. In addition, Sturm et al. reported
   that in contrast to heat-killed E. coli Nissle 1917, the viable
   bacterium might exert a more potent inhibitory effect against
   intestinal inflammation by inhibiting T cells proliferation and the
   expression of proinflammatory cytokines [[190]84].

   To investigate whether the immunomodulatory properties of SV-53 are
   mediated, in part, through epigenetic modification, DNA methylation
   analysis was conducted. In mice exposed to heat-inactivated SV-53, an
   increase in CPGs methylation around TSS of several genes controlling
   Th17 differentiation and function, including Il6, Il17rc, Il9, Il11,
   Akt1, Ikbkg, and Sgk1, was found, which may indicate transcriptional
   repression of these genes.

   As discussed earlier, IL-6 plays a central role in initiating the
   differentiation of naïve T cells into the Th17 lineage. IL-17RC is
   critical to signal transduction via IL-17A and the pathogenesis of
   autoimmune diseases. In vitro studies have shown that IL-17RC
   deficiency protects against autoimmunity [[191]85]. Moreover, it has
   been shown that Th17 cells can secrete IL-9. IL-9 neutralization and
   IL-9 receptor deficiency led to a reduction in Th17 cells and
   IL–6-producing macrophages in the central nervous system, ultimately
   ameliorating the development of experimental autoimmune
   encephalomyelitis in mice [[192]86]. IL-11 may induce Th17 cells
   generation and expansion in vitro and Th17 cells responses in vivo and
   contribute to autoimmunity. IL-11 exerts its effect on Th17 cells by
   activating the IL-6/STAT3 pathway [[193]87].

   Furthermore, Akt plays an important role in Th17 production. Upon TCR
   stimulation, the PI3k/AKT pathway contributes to the proliferation and
   survival of Th17 cells by facilitating nuclear transportation of RORγ
   and downregulating negative regulators of Th17 differentiation
   [[194]88]. Ikbkg, also known as nuclear factor-kappaB (NF-κB) essential
   modulator (NEMO) encodes the regulatory subunit of the inhibitor of
   kappaB (IkB) kinase (IKK) complex, which is essential for NF-κB
   activation [[195]89]. NF-κB contributes to the production of
   inflammatory cytokines such as IL-1, IL-6, IL-23, RORγ expression, and
   the generation of inflammatory T cells, including Th17 cells [[196]90].
   Sgk1 encodes SGK1, a serine/threonine kinase, which is downstream of
   IL-23 signaling. Sgk1 expression is specifically induced and maintained
   by exposure to IL-23. The kinase activity of SGK1 is higher in Th17
   cells compared to other subsets of T cells. On the other hand, SGK1 is
   crucial for regulating IL-23R expression, thereby maintaining the Th17
   cell phenotype by suppressing FoxO1, which negatively regulates IL-23R
   expression [[197]91].

   In addition, significant hypomethylation was detected within CPGs
   around TSS sites of Cblb and Smad4 in heat-inactivated SV-53 mice
   compared to their untreated counterparts, which may suggest the
   transcriptional activation of these genes. Cblb is a Cbl family E3
   ubiquitin ligase that restrains pathogenic Th17 cells generation and
   Th17-related autoimmunity by suppressing macrophages’ IL-6 secretion
   [[198]92]. IL-21 promotes Th17 cells differentiation and SMAD4
   negatively regulates IL-21-induced Th17 through repression of Rorc gene
   expression [[199]93]. However, we could not find any changes in the
   methylation status of genes in mice receiving live bacteria, which
   indicates that in addition to cell wall components, other components
   released during the heat-inactivation process may contribute to
   epigenetic modification by heat-inactivated SV-53.

   While results from cytokine and DNA methylation analyses may offer some
   insights into the potential role of SV-53 in regulating Th17 cells,
   flow cytometry analysis needs to be performed to elucidate the effect
   of treatment on Th17 cells production.

   In conclusion, the results of this study suggest the potential role of
   both viable and heat-inactivated probiotic SV-53, along with the
   prebiotic PCA, in improving gut immunity and homeostasis by modulating
   IgA, cytokines, and/or gut miRNAs expression. Additionally, these
   findings highlight the significant role of heat-inactivated SV-53 in
   regulating gut immunity through DNA methylation. SV-53 also enhances
   gut immunity and prevents inflammation by decreasing the population of
   pathogenic bacteria. These findings may indicate the probiotic
   characteristics of SV-53 as a novel next-generation probiotic
   bacterium.

Acknowledgments