Tetragenococcus halophilus Alleviates Intestinal Inflammation in Mice by Altering Gut Microbiota and Regulating Dendritic Cell Activation via CD83

Ulcerative colitis (UC) is one of the major subtypes of inflammatory bowel disease with unknown etiology. Probiotics have recently been introduced as a treatment for UC. Tetragenococcus halophilus (T. halophilus) is a lactic acid-producing bacterium that survives in environments with high salt concentrations, though little is known about its immunomodulatory function as a probiotic. The purpose of this study is to determine whether T. halophilus exerts an anti-inflammatory effect on intestinal inflammation in mice. Colitis was induced in C57BL/6J mice by feeding 4% DSS in drinking water for 7 days. T. halophilus was orally administered with DSS. Anti-inflammatory functions were subsequently evaluated by flow cytometry, qRT-PCT, and ELISA. Gut microbial composition was analyzed by 16S rRNA metagenomic analysis. DSS-induced colitis mice treated with T. halophilus showed less weight loss and significantly suppressed colonic shortening compared to DSS-induced colitis mice. T. halophilus significantly reduced the frequency of the dendritic cell activation molecule CD83 in peripheral blood leukocytes and intestinal epithelial lymphocytes. Frequencies of CD8+NK1.1+ cells decreased in mice with colitis after T. halophilus treatment and IL-1β levels were also reduced. Alteration of gut microbiota was observed in mice with colitis after administration of T. halophilus. These results suggest T. halophilus is effective in alleviating DSS-induced colitis in mice by altering immune regulation and gut microbiome compositions.


Introduction
Inflammatory Bowel Disease (IBD), a chronic, recurrent inflammatory disorder of the intestine, can affect any part of the gastrointestinal tract. IBD can be divided into two main types: Crohn's disease (CD) and ulcerative colitis (UC) [1]. The exact etiology of UC remains unclear. Immune dysregulation, abnormal immune response to luminal bacteria, and dysbiosis might be important factors [2]. In dextran sulfate sodium (DSS)-induced colitis models, adoptive transfer of bone marrow-derived dendritic cells (BM-DCs) can exacerbate symptoms [3], suggesting DCs play a key role in the immune balance of gut mucosa. CD83 is a glycosylated Ig-like type 1 membrane protein expressed as a surface marker for activated DCs in peripheral blood [4]. A significant number of DCs expressing CD83 molecules have been observed in CD patients [5] and murine colitis models [6]. Soluble CD83 has been suggested to have therapeutic and immunosuppressive properties by suppressing DCs-mediated T cell activation and ameliorating experimental colitis in mice [6].
Gut microbiotas play an important role in maintaining human health. They are also involved in the progression and development of human diseases [7]. UC is associated with altered compositions of the gut microbiota and mucosal immunity, resulting in excessive Table 1. Scoring of disease activity index (DAI) of DSS-induced colitis mice.

Histopathological Examination
Distal colonic sections were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 6 µm-thick sections with a microtome (Reichert-Jung Biocut 2030, Ramsey, MN, USA). These sections were then stained with hematoxylin and eosin (H&E) to analyze histological changes.

Bacterial Strain and Culture Conditions
T. halophilus was purchased from Korean Collection for Type Cultures (KCTC). For bacterial growth, T. halophilus was cultured facultative anaerobically at 30 • C for 3-4 days using an MRS medium supplemented with 6.5% NaCl.

Intestinal Epithelial Lymphocytes (IELs) Isolation
Intestinal epithelial lymphocytes (IELs) were isolated as described [15]. Mice were euthanized, and the large intestine was gently removed from the stomach using scissors; fat and connective tissues were removed, and the intestine was washed three times with ice-cold PBS to remove intestinal contents. The intestines were opened longitudinally, cut into small pieces, and transferred to an RPMI medium containing 5% DTT, 0.5M EDTA, and 2% FBS for 1 h at 37 • C. Collected samples were vortexed for 30 s and passed through a 70 µm cell strainer and then incubated with collagenase I/dispase II digestion buffer for 30 min at 37 • C. Lymphocytes were collected from the supernatant and then subjected to flow cytometry analysis.

Flow Cytometry Analysis
Leukocytes were isolated from peripheral blood (PBL). Peritoneal macrophages (pMQ) were isolated from the peritoneal cavity. IELs were isolated from the intestinal epithelium. They were then stained with anti-mouse CD40, CD83, CD80, and CD86 antibodies at 4 • C for 30 min in the dark. PBL and splenocytes were stained with CD4, CD8, CD11b, Ly6G, and NK1.1 antibodies at 4 • C for 30 min in the dark. Cells isolated from lymph nodes (LN) were stained with CD4, CD8, and NK1.1 antibodies in the dark at 4 • C for 30 min. For intracytoplasmic staining of IELs, cells were incubated with brefeldin A (10 µg/mL) in RPMI media in a 37 • C incubator supplemented with 5% CO 2 for 4 h. After incubation, cells were stained with CD4 and CD8 antibodies in the dark at 4 • C for 30 min. After washing, cells were fixed with an intracytoplasmic fixation buffer for 40 min at room temperature. After washing with a permeabilization buffer, cells were stained with interleukin (IL) -4 and IL-10 antibodies at room temperature for 40 min. The stained cells were analyzed by gating over 10,000 cells with a FACS Aria III flow cytometer (Becton Dickinson, San Jose, CA, USA). Sources of antibodies used in flow cytometry analysis are listed in Supplementary Table S1 (ST1).

Construction of 16S rRNA V3 and V4 Amplicon Sequencing Library
Fresh feces from mice were collected and analyzed for murine gut microbiota based on a 16S rRNA metagenomic sequence. In brief, the 16S rRNA gene sequencing of V3 and V4 amplicons were performed using 16S rRNA gene PCR primers (Forward Primer  5 -TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC  AG-3 , Reverse Primer 5 -GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA  GGA CTA CHV GGG TAT CTA ATC C-3 ). The libraries were sequenced on the Illumina HiSeq 2500 instrument (Illumina, St. Diego, CA, USA) (2 × 250 paired-end sequencing). Illumina adapter overhang nucleotide sequences (full-length primer sequence) were added to gene-specific sequences. At first, the adaptor sequences were removed from the original paired-end reads using CutAdapt v1.11. Secondly, the merged reads were produced with the first processed paired-end reads using FLASH v1.2.11. Then, low-quality merged reads were filtered out according to the following criteria; the read contained two or more ambiguous nucleotides, the average quality score was less than 20, and the reads' length would be shorter than 300 bp after trimming low-quality bases. Finally, the potential chimeric reads were removed using ChimeraSlayer.

Taxonomy Profiling, OTUs, Alpha-Diversity, and Beta-Diversity
To calculate taxonomic abundance, consensus sequences were clustered using the following cd-hit v4.6 parameters: identification > 99% and coverage > 80%. Consensus sequences were then aligned to the nucleotide database from NCBI using the MegaBlast algo-Cells 2022, 11, 1903 4 of 21 rithm. Finally, taxonomic profiling was performed using National Center for Biotechnology Information (NCBI) taxonomy information and KronaTools. The number of operational taxonomic units (OTU) was determined by clustering the sequence of each sample having a 97% sequence identity with cutoff using Quantitative Insights for Microbial Ecology (QIIME) software (v.1.8.0). To measure beta-diversity, the Bray-Curtis distance was applied by identifying differences between organism compositions. Principal component analysis (PCA) was then performed using beta diversity results.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Total RNA was isolated from mice PBL and splenocytes using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) following the manufacturer's recommendations. RNA was quantified with a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA) and reverse transcribed into cDNA using a PrimeScript cDNA Synthesis kit (Takara Shuzo Co., Otsu, Shiga, Japan). qRT-PCR was performed in duplicate for each target transcript using SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and gene-specific primers on a 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). For qRT-PCR, 1 µL cDNA was used as a template. The final reaction volume was 20 µL. Each reaction contained gene-specific primers listed in Supplementary Table S2 (ST2). The PCR reaction was performed with pre-denaturation at 50 • C for 2 min and 95 • C for 10 min followed by 40 cycles at 95 • C for 15 s and 60 • C for 1 min. Relative gene expression levels were calculated using the 2 −∆∆Ct method [16]. The expression level of each gene was normalized to the level of β-actin, a housekeeping gene. Data are expressed as fold change compared to untreated control.

Enzyme-Linked Immunosorbent Assay (ELISA)
After sacrifice, blood was collected from the heart of each mouse. Plasma levels of IL-1β and TNFα were analyzed using a commercial ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Mouse plasma samples were diluted 2 folds for assay. Absorbance values of samples were read at a wavelength of 450 nm using a Bio-Rad model 170-6850 microplate reader (Bio-Rad, Hercules, CA, USA). ELISA was repeated in duplicate wells.

Statistical Analysis
Statistical differences between experimental groups were determined using one-way ANOVA, followed by Bonferroni's multiple comparison test with GraphPad Prism version 8.3.1 for Windows (GraphPad Software, La Jolla, CA, USA). The Mann-Whitney U test was used to compare the differences between the two groups. Statistical significance was considered when the p-value was less than 0.05.

Administration of T. halophilus Ameliorates DSS-Induced Colitis and Reduces Disease Activity Index
Clinical symptoms of colitis over time in DSS-induced colitis mice were evaluated. These symptoms include weight loss, diarrhea, blood in stool, and death [17]. DSS-induced colitis mice had higher disease activity index (DAI) scores compared to T. halophilus treated colitis mice, indicating T. halophilus was able to prevent or ameliorate colitis symptoms in mice ( Figure 1F, Table 1). Histological examination showed extensive infiltration of Cells 2022, 11,1903 6 of 21 inflammatory cells into the colonic tissues of colitis mice ( Figure 1E). The lesions in colitis mice were reduced by treatment with T. halophilus. Although T. halophilus treated colitis mice showed some inflammatory lesions, their severity was lower compared to non-treated colitis mice.

T. halophilus Administration Affects Microbial Abundance in DSS-Induced Colitis Mice at Phylum Level
Fresh fecal samples were collected from normal control mice, DSS-induced colitis mice, and colitis mice treated with T. halophilus. V3-V4 regions of 16S rRNA of all samples were amplified. No significant differences were observed in terms of alpha diversity ( Figure 5B) or OUTs ( Figure 5A). Beta diversity analysis showed changes in species diversity and disease association along with principal components analysis (PCA) 1 and 2 ( Figure 5C). Figure 5D shows the abundance of the bacterial phylum. At the bacterial phylum level, Actinobacteria (p < 0.05) and Tenericutes (p < 0.03) showed reduced abundance in colitis mice compared to normal control mice ( Figure 5E,F), whereas Proteobacteria (p < 0.03) showed increased abundance in colitis mice compared to normal control mice ( Figure 5I). T. halophilus administration to colitis mice significantly reduced the abundance of Proteobacteria (p < 0.03, Figure 5I) and increased the population of Actinobacteria (p < 0.05, Figure 5E), Tenericutes (p < 0.03, Figure 5F), and Verrucomicrobia (p < 0.04, Figure 5G). Although Bacteroidetes and Firmicutes are the most abundant population in phyla, no significant difference was observed between the groups ( Figure 5H,K), and no significant difference in phylum Deferribacteres was observed between the groups ( Figure 5J). However, a significant difference in unassigned bacterial phylum was observed between normal control mice and non-treated colitis mice (p < 0.03, Figure 5L). The abundance of bacterial phylum is shown in Supplementary Table S3 (ST3).  Representative histograms of Ly6G+ cell frequencies in PBL and spleen are shown in (J). One-way ANOVA followed by Bonferroni's multiple comparison test was performed with GraphPad for statistical analysis. Four mice were used in each experimental group. Experiments were performed independently at least twice. Significantly different p-values are indicated by an asterisk as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

T. halophilus Administration Affects Microbial Abundance in DSS-Induced Colitis Mice at Phylum Level
Fresh fecal samples were collected from normal control mice, DSS-induced colitis mice, and colitis mice treated with T. halophilus. V3-V4 regions of 16S rRNA of all samples were amplified. No significant differences were observed in terms of alpha diversity (Figure 5B) or OUTs ( Figure 5A). Beta diversity analysis showed changes in species diversity and disease association along with principal components analysis (PCA) 1 and 2 ( Figure  5C). Figure 5D shows the abundance of the bacterial phylum. At the bacterial phylum level, Actinobacteria (p < 0.05) and Tenericutes (p < 0.03) showed reduced abundance in colitis mice compared to normal control mice ( Figure 5E,F), whereas Proteobacteria (p < 0.03) showed increased abundance in colitis mice compared to normal control mice ( Figure 5I). T. halophilus administration to colitis mice significantly reduced the abundance of Proteobacteria (p < 0.03, Figure 5I) and increased the population of Actinobacteria (p < 0.05, Figure  5E), Tenericutes (p < 0.03, Figure 5F), and Verrucomicrobia (p < 0.04, Figure 5G). Although Bacteroidetes and Firmicutes are the most abundant population in phyla, no significant difference was observed between the groups ( Figure 5H,K), and no significant difference in phylum Deferribacteres was observed between the groups ( Figure 5J). However, a significant difference in unassigned bacterial phylum was observed between normal control mice and non-treated colitis mice (p < 0.03, Figure 5L). The abundance of bacterial phylum is shown in Supplementary Table S3 (ST3). Representative histograms of Ly6G+ cell frequencies in PBL and spleen are shown in (J). One-way ANOVA followed by Bonferroni's multiple comparison test was performed with GraphPad for statistical analysis. Four mice were used in each experimental group. Experiments were performed independently at least twice. Significantly different p-values are indicated by an asterisk as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

T. halophilus Administration Downregulates IL-1β and TNFα in DSS-Induced Colitis Mice
The mRNA expression levels of NLRP3, IL-1β, and TNFα were analyzed using PBL, splenocytes, and in IELs. After T. halophilus administration to DSS-induced colitis mice, IL-1β was significantly downregulated in the spleen (p < 0.05) and in IELs (p < 0.05), respectively ( Figure 7E,H), but no significant differences were observed in NLRP3 in PBL and splenocytes among the groups ( Figure 7A,D), however, NLRP3 was significantly decreased in IELs (p < 0.05) of DSS-induced colitis mice after T. halophilus administration ( Figure 7G). In PBL, no significant differences were observed in the mRNA expression levels of NLRP3, IL-1β, and TNFα, (Figure 7A-C), but the TNFα mRNA level was significantly decreased in splenocytes (p < 0.05) after T. halophilus administration to DSS-induced colitis mice ( Figure 7F), although no significant difference was observed in IELs ( Figure 7I).  (E). A one-way ANOVA followed by Bonferroni's multiple comparison test was performed with GraphPad for statistical analysis. Four mice were used in each experimental group. Experiments were performed in duplicate. Significantly different p-values are indicated with asterisks as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

T. halophilus Administration Downregulates IL-1β and TNFα in DSS-Induced Colitis Mice
The mRNA expression levels of NLRP3, IL-1β, and TNFα were analyzed using PBL, splenocytes, and in IELs. After T. halophilus administration to DSS-induced colitis mice, IL-1β was significantly downregulated in the spleen (p < 0.05) and in IELs (p < 0.05), respectively ( Figure 7E,H), but no significant differences were observed in NLRP3 in PBL and splenocytes among the groups ( Figure 7A,D), however, NLRP3 was significantly decreased in IELs (p < 0.05) of DSS-induced colitis mice after T. halophilus administration The gating strategy of IL-4 in IELs is shown in (E). A one-way ANOVA followed by Bonferroni's multiple comparison test was performed with GraphPad for statistical analysis. Four mice were used in each experimental group. Experiments were performed in duplicate. Significantly different p-values are indicated with asterisks as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.
( Figure 7G). In PBL, no significant differences were observed in the mRNA expression levels of NLRP3, IL-1β, and TNFα, (Figure 7A-C), but the TNFα mRNA level was significantly decreased in splenocytes (p < 0.05) after T. halophilus administration to DSS-induced colitis mice ( Figure 7F), although no significant difference was observed in IELs ( Figure 7I).

T. halophilus Administration Reduces Plasma Levels of IL-1β in DSS-Induced Colitis Mice
Cytokine levels of IL-1β and TNFα in plasma samples were determined by ELISA. The IL-1β concentration was significantly higher in colitis mice compared to normal

T. halophilus Administration Reduces Plasma Levels of IL-1β in DSS-Induced Colitis Mice
Cytokine levels of IL-1β and TNFα in plasma samples were determined by ELISA. The IL-1β concentration was significantly higher in colitis mice compared to normal control mice (18.21 ± 3.01 pg/mL vs. 8.65 ± 1.54 pg/mL, p < 0.05). T. halophilus administration significantly downregulated IL-1β levels in colitis mice (7.65 ± 1.35 pg/mL vs. 18.21 ± 3.01 pg/mL, p < 0.05) compared to untreated colitis mice ( Figure 8A). TNFα levels were not different between normal control mice and colitis mice. T. halophilus administration to colitis mice reduced TNFα levels but was not statistically significant ( Figure 8B).

Discussion
We provided evidence for the ameliorative ability of T. halophilus to protect against intestinal inflammatory damage induced by DSS in colitis model mice. When DSS reaches the intestinal lumen, it acts as a toxin that destroys the integrity of the colonic mucosa, increases intestinal permeability, and exacerbates the local and systemic inflammatory response [18]. In our study, administration of T. halophilus down-regulated the disease severity index and restoration of gut microbiota. In addition, the immunomodulatory properties of T. halophilus seem to help alleviate intestinal inflammation in mice.
Administration of DSS to mice results in clinical and histopathological characteristics similar to those seen in human IBD [17]. In our study, the administration of DSS to mice leads to several clinical manifestations compared to normal mice, including weight loss, diarrhea, bloody stool, colon shorting, as well as spleen enlargement. Administration of T. halophilus reduced these clinical symptoms and alleviates colitis. T. halophilus can survive for years in up to 30% salinity during the fermentation process that makes anchovy sauce, soybean paste, red pepper paste [19], or cheese [20]. Until now, there have been few reports of the therapeutic effects of bacteria surviving in high-salinity environments such as T. halophilus in disease models. This is the first report on its anti-inflammatory function in models of inflammatory bowel diseases. The high vitality of T. halophilus, which live in high salinity and survives in bile salts and extremely low pH, can survive passing through the stomach and reach the intestine [19]. Administration of probiotics can modulate the action of DCs to produce IL-10 and IL-12 along with the expression of co-stimulatory molecules [21], and modify Th1/Th2 cytokine production in various autoimmune diseases [22].

Discussion
We provided evidence for the ameliorative ability of T. halophilus to protect against intestinal inflammatory damage induced by DSS in colitis model mice. When DSS reaches the intestinal lumen, it acts as a toxin that destroys the integrity of the colonic mucosa, increases intestinal permeability, and exacerbates the local and systemic inflammatory response [18]. In our study, administration of T. halophilus down-regulated the disease severity index and restoration of gut microbiota. In addition, the immunomodulatory properties of T. halophilus seem to help alleviate intestinal inflammation in mice.
Administration of DSS to mice results in clinical and histopathological characteristics similar to those seen in human IBD [17]. In our study, the administration of DSS to mice leads to several clinical manifestations compared to normal mice, including weight loss, diarrhea, bloody stool, colon shorting, as well as spleen enlargement. Administration of T. halophilus reduced these clinical symptoms and alleviates colitis. T. halophilus can survive for years in up to 30% salinity during the fermentation process that makes anchovy sauce, soybean paste, red pepper paste [19], or cheese [20]. Until now, there have been few reports of the therapeutic effects of bacteria surviving in high-salinity environments such as T. halophilus in disease models. This is the first report on its anti-inflammatory function in models of inflammatory bowel diseases. The high vitality of T. halophilus, which live in high salinity and survives in bile salts and extremely low pH, can survive passing through the stomach and reach the intestine [19]. Administration of probiotics can modulate the action of DCs to produce IL-10 and IL-12 along with the expression of co-stimulatory molecules [21], and modify Th1/Th2 cytokine production in various autoimmune diseases [22].
According to Grosche et al., CD83 plays an important role in controlling and maintaining immune tolerance [23]. In IBD patients, accidental activation of CD83 by microbial antigen induces Th1 and Th17 cell's immune responses in intestinal tissue. This response is characterized by the release of excessive amounts of pro-inflammatory cytokines along with the activation of macrophages and granulocytes that promote inflammation [24]. Downregulation of CD83 by T. halophilus administration can be explained by bacterial specificity. According to Amar et al., it was reported that the expression levels of CD80 and CD83 differ depending on the type of bacteria [25]. The regulation of CD83 and CD80 appears to be more affected by T. halophilus than other costimulatory molecules. CD83 is known to be expressed at higher levels in UC patients [26], and our present study shows inhibition of CD83 through administration of T. halophilus and amelioration of inflammation are correlated.
It has also been reported T. halophilus significantly augmented the gene expressions of IL-10 [13], and IL-10 deficient mice have been used as a model reflecting the characteristics of colitis [27]. Increased IL-10 secretion reduces pro-inflammatory cytokines and prevents cell damage [28]. T. halophilus Strain Th221 provided a therapeutic effect in patients with perennial allergic rhinitis by inhibiting IgE production after oral ingestion in the heat-inactivated form [29,30]. The action mechanism heat-inactivated form may be different with live bacterial administration. Xiong et al. demonstrated that the combination treatment of IL-4 and IL-10 significantly inhibited TNBS-induced colon tissue damage in mice [31]. IL-4 and IL-10 have anti-inflammatory and immunoregulatory effects and play an important role in the mucosal immune system by inhibiting the synthesis of proinflammatory cytokines that alleviate intestinal inflammation [32][33][34][35]. Colitis mice treated with Trichinella spiralis extracellular vesicles (Ts-Evs) showed a significant increase in the CD4+IL-4+ cell populations compared to the TNBS group, which was associated with ameliorating colitis by the expansion of Th2 cells [36]. IL-10 inhibits IFN-γ production by Th1 cells and reduces Th17 responses in the DSS model [37][38][39], and mice with IL-10-deficient Treg develop spontaneous colitis [40,41]. In our study, the decrease in IL-4+ and IL-10+ cell frequency in colitis mouse IELs was restored by the administration of T. halophilus, demonstrating that inflammation-related cells invading intestinal tissues may be regulated by T. halophilus.
Fecal microbiota transplantation has an ameliorative role in UC patients [42], suggesting that microbiota plays a role in the disease. Attenuation of inflammation induced by T. halophilus was further validated in the stool microbiome. Akkermansia muciniphilia (A. muciniphilia), a microbial species identified as a major mucin decomposer of the gut microbiota, was reduced in UC patients [43], and administration of Lactobacillus reuteri (L. reuteri) to IBD patients may reduce inflammation [44]. In our study, an abundance of A. muciniphilia and L. reuteri was decreased in DSS-induced colitis mice more than in normal mice, which recovered by T. halophilus treatment. In addition, L. johnsonni and Intestinimonas butyriciproducens were depleted in colitis mice and corrected after treatment with T. halophilus. Previous studies have reported significant reductions in genera Alistipes, Barnesiella, Oscillibacter, and Ruminococcus observed in IBD [45]. With the same trend, we found Alistipes, Barnesiella, and Ruminococcus were reduced in colitis mice and recovered after administration of T. halophilus. Lactobacillus performs anti-inflammatory properties by breaking down proinflammatory cytokines [46], and Bifidobacterium has an anti-inflammatory function through modification of the SOCS gene [47]. In our study, administration of T. halophilus to colitis mice increased the abundance of Lactobacillus and Bifidobacterium.
CD1d independent NK1.1+CD8+ T cells produced high levels of IFN-γ and TNFα, contributing to colitis pathogenesis, and suggested that DCs and macrophages might be responsible for increasing NK1.1+CD8+ T cells through IL-15 [48]. In active UC, neutrophils have been observed in the crypt epithelium and lamina propria of colonic tissues [49,50], suggesting that neutrophils might play an important role in the pathogenesis of UC. Neutrophil depletion with an anti-Ly6G antibody can ameliorate DSS-induced colitis in rats [51,52]. In this present study, administration of T. halophilus affected the frequency of CD11b+Ly6G+ cells in colitis mice and probably had a positive effect on the improvement of intestinal inflammation. IL-1β is a pro-inflammatory cytokine that plays an integral role in IBD [53,54]. Administration of IL-1β to germ-free mice develops Th17 cells in the small intestine [55], in addition, IL-1β production by commensal bacteria can promote intestinal inflammation [56]. IL-1β was found to be upregulated in active UC patients [57], which activates neutrophils and promotes intestinal pathology [58,59]. Serum concentrations of proinflammatory cytokine TNFα were not different between patients with active or inactive CD or UC, suggesting serum TNFα is not an adequate biomarker for assessing the disease activity in patients with IBD [60]. However, some studies have found that TNFα levels were increased in UC patients and anti-TNFα therapy has promising therapeutic effects [54]. In our study, T. halophilus suppresses serum IL-1β and TNFα levels in DSS-induced colitis mice.
Administration of T. halophilus may alleviate acute colitis symptoms in mice with DSSinduced colitis, but these experiments were performed in mice fed a standardized diet and the small number of experimental groups may be a limitation of the study. Nevertheless, the current study provides in vivo evidence T. halophilus has favorable outcomes against DSS-induced colitis in mice, which may serve as a method for gut microbiome control to complement current therapies for colitis. The anti-inflammatory effects on DSS-induced colitis through the remodeling of the gut microbiota may have been partially immunomodulatory, and therefore further studies are needed to evaluate the immunomodulatory functions of T. halophilus in colitis on a large scale.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/cells11121903/s1, Figure S1: Effects of T. halophilus on activation of dendritic cells in peritoneal macrophage (pMQ) of DSS-induced colitis mice. Figure S2: Frequencies of CD4+ cells in peripheral blood leukocytes (PBL) and spleens of normal control mice, normal mice treated with T. halophilus, DSS-induced colitis mice, and colitis mice treated with T. halophilus. Figure S3: Frequencies of CD4+, CD8+, NK1.1+, and CD8+NK1.1+ cells in lymph nodes (LN) of normal control mice, normal mice treated with T. halophilus, DSS-induced colitis mice, and colitis mice treated with T. halophilus. Table S1: Sources of antibodies used in this work. Table S2: List of primers used in real-time PCR. Table S3: Identification of differences in gut microbiota between normal mice, DSS-induced colitis mice, and T. halophilus-treated colitis mice.

Institutional Review Board Statement:
All animal studies were carried out in accordance with the guidelines for the care and use of laboratory animals after obtaining approval from Ajou University Institutional Animal Care and Use Committee (approval number: AMC-2021-0060).

Informed Consent Statement: Not applicable.
Data Availability Statement: The16S rRNA metagenomic data has been deposited in NCBI under the accession number PRJNA772551.