Modulatory Effects of Heat-Inactivated Streptococcus Thermophilus Strain 7 on the Inflammatory Response: A Study on an Animal Model with TLR3-Induced Intestinal Injury

Rotavirus infections result in severe gastroenteritis with a detrimental inflammatory response in the intestine. Because probiotics have an anti-inflammatory effect and can modulate the gut microbiota profile, they can be used as a biotherapy for inflammatory intestinal diseases. In this study, we isolated Streptococcus thermophilus strain 7 (ST7) from cow milk and examined the effect of heat-inactivated ST7 on the intestinal histopathological score, inflammatory cytokine levels, T-cell activation and effector function, and microbiome profile in a mouse model with intestinal injury induced by polyinosinic-polycytidylic acid (poly I:C), a Toll-like receptor 3 agonist. The results indicated that ST7 treatment prevented weight loss and intestinal injury and prevented the upregulation of serum interleukin-6 (IL-6), tumor necrosis factor-α, and IL-15 levels in intestinal epithelial cells; prevented the upregulation of inflammation-associated Gammaproteobacteria and Alistipes; and increased the levels of Firmicutes in fecal microbiota after poly I:C stimulation. ST7 treatment also increased the serum interferon-γ (IFN-γ) level and promoted the expression of IFN-γ in both CD8 and CD4 T cells. In summary, ST7 prevented the inflammatory response, promoted the T-cell effector function, and modulated the microbiota profile of mice with poly I:C-induced small intestine injury.


Introduction
Rotavirus infections are a leading cause of severe gastroenteritis in children [1]. Rotaviruses are double-stranded RNA (dsRNA) viruses that stimulate inflammatory responses in the intestine [2,3] and alter the function of the small intestine epithelium, resulting in diarrhea. The destruction of enterocytes results in primary malabsorption, decreased digestion, and acute diarrhea [3]. Enterocytes distinguish microbial antigens through pattern recognition receptors, such as Toll-like receptors (TLRs), which play a critical role in innate immunity by facilitating the identification of the genomic dsRNA of several viruses or dsRNA synthesized during viral replication. Polyinosinic-polycytidylic acid (poly I:C) is a synthetic analog of retroviral genomic dsRNA. It stimulates TLR3, which in turn activates nuclear factor-κB and induces the production of type I interferon (IFN) [4]. In a poly I:C-induced intestinal injury animal model, the intraperitoneal administration of poly I:C triggered a local intestinal immune response and caused severe mucosal damage in the gut in a TLR3-dependent manner, and, neutralizing the effects of interleukin-15 (IL-15),

Intestinal Histopathological Evaluation
Small intestine samples were excised, washed with PBS, and immersed in a formalin solution. Once fixed, the samples were dehydrated, embedded, cut into 4 µm thick serial sections, and stained with hematoxylin and eosin (H&E) for examination under a light microscope. All slides were coded and evaluated in a blinded manner in sections. Histopathologic scores and villus lengths were evaluated as described previously [21,22]. A summary of histopathologic scores was indicated by the following description. Score 0: normal ileum with intact epithelium; Score 1: mild mucosal inflammatory cell infiltrate; Score 2: mild diffuse inflammatory cell infiltrate in mucosa and submucosa; Score 3: moderate inflammatory cell infiltrates in mucosa and submucosa with villous blunting.

Immunohistochemistry
To perform an immunohistochemical analysis, intestine samples were fixed in formalin for 3 days, and 0.5 cm coronal slices were embedded in paraffin and cut into 4 µm thick sections [23,24]. The sections were then deparaffinized, rehydrated, and subjected to an antigen retrieval process and then stained with an anti-IL-15 antibody (R&D Systems, Minneapolis, MN, USA), followed by incubation with a peroxidase-conjugated AffiniPure Donkey Anti-Goat IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Subsequently, the sections were stained with 3,3 -diaminobenzidine and hematoxylin (BioLegend, San Diego, CA, USA) and were observed under a microscope (Olympus BX43; Olympus, Tokyo, Japan). IL-15-positive areas were quantified using HistoQuest tissue analysis software (TissueGnostics, Vienna, Austria).

Serum Cytokine Concentrations
Serum TNF-α, IL-6, and IFN-γ concentrations were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits in accordance with the manufacturer's recommendations (BioLegend and R&D Systems). The range of the standard curves was between 7.8 and 500 pg/mL.

16S Ribosomal Ribonucleic Acid Gene and Next-Generation Sequencing
The detailed procedures for the high-throughput sequencing of the 16S ribosomal ribonucleic acid (rRNA) gene and next-generation sequencing are described in our pre-vious study [25]. The mice were allowed to defecate freely in clean cages, and DNA was extracted from fresh stool samples by using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). Library preparation was conducted using an Illumina MiSeq system in accordance with the protocol for 16S rRNA gene amplicons. Universal primers (341F and 805R) were used to amplify the V3-V4 region of the bacterial 16S rRNA genes. Demultiplexed, paired reads were removed using Cutadapt (v1.12). Filtered reads were processed using the DADA2 package (v1.14.1) in R software (v3.6.1) [26,27]. However, the rarefying procedure was not performed. The V3-V4 sequence variants in the samples were inferred using the DADA2 package, and the frequency of each sequence variant in each sample was determined. The taxonomy assignment was conducted using the SILVA database (v138) [28], with a minimum bootstrap confidence of 80. The multiple sequence alignment of variants and phylogenetic tree preparation were performed using DECIPHER (v2.14.0) and the phangorn package (v2.5.5), respectively [29]. Taxonomy assignment, count tables, and phylogenetic trees were applied to a phyloseq object, and community analysis was performed using phyloseq software (v1.30.0) [30]. Alpha diversity indices were calculated to estimate the richness function of the phyloseq package. All statistical analyses were performed using the Wilcoxon-Mann-Whitney test (α = 0.05). To assess community dissimilarity between the groups, UniFrac distances were calculated using the GUniFrac package (v1.1) [31]. Principal coordinate analysis (PCoA) ordination was applied for UniFrac distances, and the adonis and betadisper functions from the vegan package (v2.4) of R were used to analyze the dissimilarity of compositions between the groups and the homogeneity of their dispersion, respectively.

Flow Cytometry
Spleens and mesenteric lymph nodes were collected from the control, poly I:C-treated, ST7-treated, and polyI:C+ST7-treated mice. All tissues were ground using a Wheaton tissue grinder, and then a cell strainer was used to filter out the debris. Red blood cells were lysed in an RBC lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). Splenocytes and lymph node cells were then suspended in a Roswell Park Memorial Institute (RPMI) culture medium containing 2% fetal bovine serum. To minimize the nonspecific binding of antibodies to FcR-bearing cells, a 2.4G2 hybridoma supernatant was used to pretreat these splenocytes and lymph node cells. After the 2.4G2 supernatant was removed by centrifugation, the cell surface molecules were stained with specific antibodies for 10 min and then washed twice with a staining buffer (1× PBS, 2% horse serum, and 0.05% NaN 3 ). Finally, all cells were resuspended in a staining buffer and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). The following antibodies were used in conjugation with a fluorochrome from BioLegend: CD3-FITC (2C11), CD8α-APC-Cy7 (53-6.7), CD19-PB (6D5), CD4-PE-Cy7 (GK1.5), and CD69-PE (H1.2F3). Positive cells were validated through comparison with the no-antibody staining control.

In Vitro Splenocyte Stimulation Assay
Splenocytes were prepared as described in Section 2.7. Cells were suspended in an RPMI culture medium containing 10% fetal bovine serum. The trypan blue exclusion assay was used to count the number of viable splenocytes. Subsequently, 5 × 10 5 viable splenocytes were stimulated with ST7 (5 × 10 4 U/mL), poly I:C (100 µg/mL), or both (poly I:C+ST7) in a 96-well plate containing culture medium for 24 h. The culture supernatant was then collected, and the IFN-γ level was determined using ELISA in accordance with standard protocols.

Intracellular IFN-γ Detection Assay
A total of 2 × 10 6 splenocytes were incubated with phorbol myristate acetate (100 ng/mL) and an ionophore (A23187, 1 µg/mL) in a 0.5 mL RPMI culture medium containing 10% fetal bovine serum for 3 h. Brefeldin A (5 µg/mL) was added at the last 2 h of culture. The cells were first stained with antibodies for T-cell-related surface markers and Fixable Viability Dye eFluor 506 (Thermo Fisher Scientific) and then fixed by a Foxp3 Fix/Perm Buffer Set in accordance with standard protocols (BioLegend). Finally, the cells were intracellularly stained with an IFN-γ-APC (XMG1.2) antibody or an isotype control antibody for 20 min and were analyzed using flow cytometry.

Statistical Analysis
For data comparison, Mann-Whitney t tests and one-way analysis of variance were conducted using GraphPad Prism software (GraphPad, La Jolla, CA, USA). The error bars in the results represent the standard errors of the mean. Microbiota enrichment analysis was conducted using the linear discriminant analysis (LDA) effect size method. Data were compared using the Kruskal-Wallis and Wilcoxon tests. Differences were regarded as statistically significant at p ≤ 0.05, with a logarithmic LDA score of ≥2 [32].

Effects of ST7 Treatment on Poly I:C-Induced Intestinal Damage and Weight Loss
To determine the overall health condition and intestinal alterations following the administration of poly I:C, changes in weight and intestinal histology were evaluated. The results revealed that the administration of poly I:C resulted in a considerable reduction in weight ( Figure 1B) and induced intestinal tissue inflammation with mild mucosal erosion ( Figure 1C). ST7 treatment also considerably prevented weight loss, improved the histopathological score of the intestine ( Figure 1B,C), and prevented intestinal villus length reductions induced by poly I:C stimulation. Taken together, these results revealed that ST7 treatment prevented TLR3-induced intestinal damage.

Effects of ST7 Treatment on the Serum Cytokine Profile after Poly I:C Stimulation
We next evaluated the systemic inflammation level by determining the serum inflammatory cytokine levels. The intraperitoneal administration of poly I:C increased serum IL-6 and TNF-α levels ( Figure 2), and these levels were substantially low in the ST7 treatment group (poly I:C + ST7). IFN-γ levels were also considerably increased in the poly I:C The mice received ST7 orally for 7 days. On day 8, they were intraperitoneally injected with poly I:C for 2 h, and then their sera, spleens, and lymph nodes were harvested to examine the cytokine and immune cell activation profiles. Data pertaining to weight, microbiota, and intestinal histology were collected 24 h after poly I:C stimulation. (B) Quantification of the percentage of weight loss in mice treated with poly I:C and poly I:C + ST7. n = 5, * p < 0.05. **** p < 0.0001. (C) Representative photographs of H&E-stained paraffin-embedded sections from mice treated with poly I:C and poly I:C + ST7. The histopathological scores and villus-to-crypt ratios were quantified in the graph. n = 5, * p < 0.05, ** p < 0.01.

Effects of ST7 Treatment on the Serum Cytokine Profile after Poly I:C Stimulation
We next evaluated the systemic inflammation level by determining the serum inflammatory cytokine levels. The intraperitoneal administration of poly I:C increased serum IL-6 and TNF-α levels ( Figure 2), and these levels were substantially low in the ST7 treatment group (poly I:C + ST7). IFN-γ levels were also considerably increased in the poly I:C group. Notably, ST7 treatment synergistically increased the serum IFN-γ level ( Figure 2). their sera, spleens, and lymph nodes were harvested to examine the cytokine and immune ce vation profiles. Data pertaining to weight, microbiota, and intestinal histology were collecte after poly I:C stimulation. (B) Quantification of the percentage of weight loss in mice treated poly I:C and poly I:C + ST7. n = 5, * p < 0.05. **** p < 0.0001. (C) Representative photographs of stained paraffin-embedded sections from mice treated with poly I:C and poly I:C + ST7. The pathological scores and villus-to-crypt ratios were quantified in the graph. n = 5, * p < 0.05, 0.01.

Effects of ST7 Treatment on the Serum Cytokine Profile after Poly I:C Stimulation
We next evaluated the systemic inflammation level by determining the serum in matory cytokine levels. The intraperitoneal administration of poly I:C increased seru 6 and TNF-α levels (Figure 2), and these levels were substantially low in the ST7 treat group (poly I:C + ST7). IFN-γ levels were also considerably increased in the pol group. Notably, ST7 treatment synergistically increased the serum IFN-γ level (Figu Serum samples were collected from control (PBS-treated), poly I:C-treated, and poly I:C+ST7-treated mice. Serum IL-6, TNF-α, and IFN-γ levels were measured after poly I:C stimulation for 2 h. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Effects of ST7 Treatment on Intestinal IL-15 Expression in Poly I:C-Treated Mice
To determine how ST7 treatment prevented poly I:C-induced intestinal injury, we examined the expression of IL-15 in the intestine by using an immunohistochemical assay. Generally, IEC-derived IL-15 promotes the poly I:C-enhanced cytotoxicity of intraepithelial lymphocytes (IELs), which in turn results in intestinal injury [5]. Our data indicated that poly I:C considerably increased the expression of IL-15 in the IECs of the intestinal villi. In addition, ST7 treatment substantially prevented the upregulation of poly I:C-stimulated IL-15 expression in IECs ( Figure 3).

Effects of ST7 Treatment on Intestinal IL-15 Expression in Poly I:C-Treated Mice
To determine how ST7 treatment prevented poly I:C-induced intestinal injury, we examined the expression of IL-15 in the intestine by using an immunohistochemical assay. Generally, IEC-derived IL-15 promotes the poly I:C-enhanced cytotoxicity of intraepithelial lymphocytes (IELs), which in turn results in intestinal injury [5]. Our data indicated that poly I:C considerably increased the expression of IL-15 in the IECs of the intestinal villi. In addition, ST7 treatment substantially prevented the upregulation of poly I:C-stimulated IL-15 expression in IECs ( Figure 3). Inflammation and infection are accompanied by gut dysbiosis [33]. In this study, we examined whether ST7 can modulate the gut microbiota profile induced by poly I:C-induced inflammation. The results indicated that the alpha diversity of the fecal microbiota in the poly I:C treatment group was similar to that in the control group ( Figure 4A). Eval-

Effects of ST7 Treatment on the Microbiota Profile of Poly I:C-Treated Mice
Inflammation and infection are accompanied by gut dysbiosis [33]. In this study, we examined whether ST7 can modulate the gut microbiota profile induced by poly I:Cinduced inflammation. The results indicated that the alpha diversity of the fecal microbiota in the poly I:C treatment group was similar to that in the control group ( Figure 4A). Evaluation of beta diversity through PCoA in variance-adjusted weighted UniFrac analysis revealed that, compared with the control group, the poly I:C treatment group had a considerably altered fecal microbiota profile ( Figure 5B, left panel). Figure 5A depicts the substantial between-group differences in the abundance of microbiota species. Compared with the poly I:C treatment group, the control group had considerably higher LDA scores for Actinobacteriota, Bacteroidota, Campylobacterota, and Patescibacteria at the phylum level. In addition, compared with the control group, the poly I:C treatment group had higher LDA scores for Proteobacteria at the phylum level, Gammaproteobacteria at the class level, and Alistipes at the genus level. After 1 week of ST7 treatment and subsequent poly I:C stimulation, the fecal microbiota profile was substantially different from that of the poly I:C treatment group ( Figure 4B, right panel). However, no considerable difference was detected in alpha diversity ( Figure 4A). The LDA scores for Firmicutes at the phylum level, Clostridia at the class level, and Lachnospirales at the order level in the mice that received ST7 treatment and poly I:C stimulation (poly I:C + ST7) were higher than those of the poly I:C-treated control group ( Figure 5A). In an inflammatory intestinal disease, a decreased Firmicutes-to-Bacteroidetes ratio is observed [34]. In this study, this ratio was considerably increased by ST7 treatment in the poly I:C treatment group ( Figure 5C). Taken together, these results indicate that ST7 treatment can ameliorate dysbiosis in mice with poly I:C-induced intestinal injury. level, Clostridia at the class level, and Lachnospirales at the order level in the mice that received ST7 treatment and poly I:C stimulation (poly I:C + ST7) were higher than those of the poly I:C-treated control group ( Figure 5A). In an inflammatory intestinal disease, a decreased Firmicutes-to-Bacteroidetes ratio is observed [34]. In this study, this ratio was considerably increased by ST7 treatment in the poly I:C treatment group ( Figure 5C). Taken together, these results indicate that ST7 treatment can ameliorate dysbiosis in mice with poly I:C-induced intestinal injury.

Effects of ST7 Treatment on T-Cell Activation after Poly I:C Stimulation
We next investigated whether ST7 treatment modulates T-cell activation by examining CD69 expression after poly I:C stimulation. The results revealed that neither poly I:C stimulation nor ST7 treatment affected the percentages of CD69 + CD8 + T and CD69 + CD4 + T cells in the mesenteric lymph nodes. However, after ST7 treatment for 7 days, the percentages of CD69 + CD8 + T and CD69 + CD4 + T cells increased after 2 h of poly I:C stimulation ( Figure 6A). In the spleen, poly I:C stimulation considerably increased the percentage of CD69 + CD8 + T cells and slightly increased the percentage of CD69 + CD4 + T-cells ( Figure 6B). ST7 treatment also considerably increased the percentages of CD69 + CD8 + and CD69 + CD4 + T cells in the spleen after 2 h of poly I:C stimulation ( Figure 6B). These results indicated that ST7 treatment can promote T-cell activation upon TLR3 activation.

Effects of ST7 Treatment on T-Cell Activation after Poly I:C Stimulation
We next investigated whether ST7 treatment modulates T-cell activation by examining CD69 expression after poly I:C stimulation. The results revealed that neither poly I:C stimulation nor ST7 treatment affected the percentages of CD69 + CD8 + T and CD69 + CD4 + T cells in the mesenteric lymph nodes. However, after ST7 treatment for 7 days, the percentages of CD69 + CD8 + T and CD69 + CD4 + T cells increased after 2 h of poly I:C stimulation ( Figure 6A). In the spleen, poly I:C stimulation considerably increased the percentage of CD69 + CD8 + T cells and slightly increased the percentage of CD69 + CD4 + Tcells ( Figure 6B). ST7 treatment also considerably increased the percentages of CD69 + CD8 + and CD69 + CD4 + T cells in the spleen after 2 h of poly I:C stimulation ( Figure 6B). These results indicated that ST7 treatment can promote T-cell activation upon TLR3 activation.

Effects of ST7 Treatment on T-Cell Activation and IFN-γ Production after Poly I:C Stimulation
IFN-γ is a critical effector cytokine that induces an antiviral immune response. In this study, because ST7 treatment increased the serum IFN-γ level upon TLR3 activation, we quantified IFN-γ levels in T cells after poly I:C stimulation. In vitro splenocyte stimulation revealed that ST7 significantly increased the production of IFN-γ from splenocytes upon poly I:C stimulation ( Figure 7A). In addition, the results of the oral heat-inactivated ST7 experiment revealed that in vivo poly I:C stimulation for 2 h did not affect IFN-γ levels in CD8 + T and CD4 + T cells. ST7 treatment for 7 days led to the upregulation of IFN-γ expression in splenic CD8 and CD4 T cells not only directly ( Figure 7B, control vs. ST7) but also upon poly I:C stimulation ( Figure 7B, poly I:C vs. poly I:C + ST7). Taken together, these results indicated that ST7 treatment can promote T-cell effector function.

Effects of ST7 Treatment on T-Cell Activation and IFN-γ Production after Poly I:C Stimulation
IFN-γ is a critical effector cytokine that induces an antiviral immune response. In this study, because ST7 treatment increased the serum IFN-γ level upon TLR3 activation, we quantified IFN-γ levels in T cells after poly I:C stimulation. In vitro splenocyte stimulation revealed that ST7 significantly increased the production of IFN-γ from splenocytes upon poly I:C stimulation ( Figure 7A). In addition, the results of the oral heat-inactivated ST7 experiment revealed that in vivo poly I:C stimulation for 2 h did not affect IFN-γ levels in CD8 + T and CD4 + T cells. ST7 treatment for 7 days led to the upregulation of IFN-γ expression in splenic CD8 and CD4 T cells not only directly ( Figure 7B, control vs. ST7) but also upon poly I:C stimulation ( Figure 7B, poly I:C vs. poly I:C + ST7). Taken together, these results indicated that ST7 treatment can promote T-cell effector function.

Discussion
The heat-inactivated S. thermophilus possess an immunomodulatory function. In this study, we discovered that, in a poly I:C-induced intestinal damage mouse model, heatinactivated ST7 treatment modulated the host fecal microbiota and inflammatory response. The results indicated that oral ST7 treatment effectively prevented poly I:C-induced injury to the intestinal epithelium by preventing the upregulation of serum inflammatory cytokines (TNF-α and IL-6) and IL-15 in IECs. In addition, ST7 treatment enhanced host antiviral activity by enhancing T-cell activation and effector function under poly I:C stimulation conditions. It also substantially altered the gut microbiota profile; that is, it prevented the upregulation of inflammation-associated Gammaproteobacteria and Alistipes and increased the levels of Firmicutes in the fecal microbiota after poly I:C stimulation. Taken together, our findings indicated that ST7 prevented the inflammatory response, enhanced the T-cell effector function, and modulated the microbiota profile of mice with poly I:C-induced intestinal injury.
Intestinal inflammation and infection are accompanied by an imbalance in the gut microbiota [33]. Viral infections elicit an inflammatory response, resulting in gut barrier dysfunction [35]. However, the mechanism through which a dsRNA virus infection affects the gut microbiome remains unclear. In this study, to mimic a dsRNA viral infection, we used intraperitoneal poly I:C injections, which resulted in substantial gut microbial dysbiosis and increased the number of Gammaproteobacteria and Alistipes in the fecal microbiota; these alterations are associated with inflammatory responses in colitis and hypertension, respectively [36,37]. Notably, the numbers of Gammaproteobacteria in the ST7-treated mice were not higher than those in the poly I:C-treated mice. ST7 treatment considerably increased the numbers of Firmicutes and Clostridium species in the poly I:Ctreated mice. Firmicutes have anti-inflammatory effects and can alleviate the progression

Discussion
The heat-inactivated S. thermophilus possess an immunomodulatory function. In this study, we discovered that, in a poly I:C-induced intestinal damage mouse model, heatinactivated ST7 treatment modulated the host fecal microbiota and inflammatory response. The results indicated that oral ST7 treatment effectively prevented poly I:C-induced injury to the intestinal epithelium by preventing the upregulation of serum inflammatory cytokines (TNF-α and IL-6) and IL-15 in IECs. In addition, ST7 treatment enhanced host antiviral activity by enhancing T-cell activation and effector function under poly I:C stimulation conditions. It also substantially altered the gut microbiota profile; that is, it prevented the upregulation of inflammation-associated Gammaproteobacteria and Alistipes and increased the levels of Firmicutes in the fecal microbiota after poly I:C stimulation. Taken together, our findings indicated that ST7 prevented the inflammatory response, enhanced the T-cell effector function, and modulated the microbiota profile of mice with poly I:C-induced intestinal injury.
Intestinal inflammation and infection are accompanied by an imbalance in the gut microbiota [33]. Viral infections elicit an inflammatory response, resulting in gut barrier dysfunction [35]. However, the mechanism through which a dsRNA virus infection affects the gut microbiome remains unclear. In this study, to mimic a dsRNA viral infection, we used intraperitoneal poly I:C injections, which resulted in substantial gut microbial dysbiosis and increased the number of Gammaproteobacteria and Alistipes in the fecal microbiota; these alterations are associated with inflammatory responses in colitis and hypertension, respectively [36,37]. Notably, the numbers of Gammaproteobacteria in the ST7-treated mice were not higher than those in the poly I:C-treated mice. ST7 treatment considerably increased the numbers of Firmicutes and Clostridium species in the poly I:C-treated mice. Firmicutes have anti-inflammatory effects and can alleviate the progression of inflamma-tory bowel disease [34]. The Clostridium species positively regulates the differentiation, function, and accumulation of T cells in the mouse colon, providing anti-inflammatory and protective effects [38]. Collectively, our data suggest that the oral administration of heatinactivated ST7 can prevent the dysbiosis of microbiota by modulating the proinflammatory microbiota profile toward anti-inflammatory properties upon dsRNA viral stimulation. According to a previous study, the intestinal dysbiosis profile of a TLR4-induced sepsis animal model differs from that of a TLR3-induced intestinal injury model [16]. In addition, intraperitoneal LPS treatment considerably decreases the numbers of Fusobacterium in the fecal microbiota, and oral live S. thermophiles treatment considerably increases the numbers of Fusobacterium after the administration of LPS [16]. However, in this study, the numbers of Fusobacterium were not affected by either poly I:C or poly I:C + ST7 treatment. Taken together, these data indicate that S. thermophiles can modulate different types of TLR-induced microbial dysbiosis.
Strains of S. thermophilus can modulate immune responses, with each strain exerting either a proinflammatory or an anti-inflammatory effect [16,39]. In a TLR4-induced sepsis animal model, treatment with S. thermophiles alleviates intestinal injury and decreases the serum levels of inflammatory cytokines (IL-6 and TNF-α) [16]. This finding is in line with that of our study, in which we reported that S. thermophilus bacteria exhibit an antiinflammatory property. Although ST7 treatment increased serum IFN-γ amounts and IFN-γ can potentiate pro-inflammatory signaling [40], ST7 can differentially affect inflammatory cytokines (IL-6 and TNF-α) and IFN-γ production after 2 h poly I:C stimulation. Upon poly I:C stimulation, oral ST7 treatment prevents IL-15 overexpression in the IECs, thereby potentially protecting the intestine from the detrimental poly I:C-induced immune response. After 2 h of poly I:C stimulation, ST7 treatment enhances not only the activation of CD8 and CD4 T cells but also their IFN-γ production. IFN-γ can promote the type I interferons, including IFN-α and IFN-β, which can antagonize virus replication [9,41]. These findings suggest that ST7 enhances T-cell effector function during the early stage of viral infection. Taken together, these findings imply that ST7 can prevent TLR3-induced inflammation and enhance the T-cell immune response during the early stage of viral infection.
Numerous studies have indicated that both viable and heat-inactivated lactic acid bacteria can modulate the microbiome. For instance, gut microbial co-occurrence network analysis has revealed that live and heat-inactivated Lactiplantibacillus plantarum can reorganize human gut microbial community structures [42]. Another animal study demonstrated that viable and heat-inactivated Lactobacillus plantarum can reduce hypercholesterolemia and regulate the intestinal microbiota and metabolites related to lipid metabolism [43]. In addition, various strains of heat-killed lactic acid bacteria (S. thermophilus, L. bulgaricus, and L. acidophilus) can disrupt the expression of the tight junction proteins ZO-1 and occludin and prevent epithelial barrier dysfunction induced by inflammatory cytokines [44]. However, although live S. thermophilus can modulate intestinal dysbiosis in mice with sepsis [16], whether and how nonviable S. thermophilus modulates the intestinal microbiome remain unclear. In this study, heat-inactivated ST7 pretreatment prevented epithelial barrier dysfunction and prevented the increase in TNF-α levels in the serum after poly I:C stimulation. This anti-inflammatory effect can protect IECs against inflammatory-cytokine-induced dysfunction and IL-15-induced IEC and IEL cytotoxicity [45]. However, the mechanism through which ST7 functions remains unclear and warrants further investigation.

Conclusions
Heat-inactivated ST7 treatment has an immunomodulatory property, prevents detrimental inflammatory responses in the intestine, and modulates gut microbial dysbiosis. ST7 treatment also prevents the upregulation of inflammatory cytokines (IL-6 and TNF-α) in the serum and the expression of IL-15 in the IECs and promotes the activation of T cells and their IFN-γ production. In addition, ST7 treatment prevents the upregulation of inflammation-associated Gammaproteobacteria and Alistipes and increases the numbers of Firmicutes in the fecal microbiota after poly I:C stimulation. This finding indicates that heated-inactivated ST7 can be used to prevent injuries resulting from dsRNA-virus-induced intestinal inflammation, which in turn may prevent intestinal dysfunction due to gastroenteritis. Regarding the limitations of this study, the biological components of heat-inactivated ST7 that are responsible for these anti-inflammatory and immune modulation activities remain unclear, and the underlying mechanisms require extensive investigations.  Data Availability Statement: The data sets used and/or analyzed in this study are available from the corresponding authors upon reasonable request.