Oral fermented rice bran supplementation suppresses viral replication and stimulates immune functions in immunocompetent and immunocompromised mice infected with influenza virus

Abstract

Background

The number of immunosuppressed elderly persons, who are at a higher risk of developing influenza infection-related complications, has been increasing. Fermented rice bran (FRB) is used as a dietary supplement, exhibits a variety of biological activities, and may exhibit anti-influenza virus activity. However, the effect of FRB supplements on influenza A virus (IFV) infection remains unknown. We aimed to investigate the effects of FRB on influenza prevention by oral administration to IFV-infected mice in an immunocompetent or immunocompromised state.

Methods

FRB was produced by fermentation of rice bran with a mixture of Bacillus spp, Lactobacillus sp, Bifidobacterium sp, and Aspergillus sp. BALB/c mice orally received FRB (20 mg/day) for 1 or 2 months prior to intranasal IFV inoculation or immediately after IFV infection until 14 days post-inoculation. Weight changes, virus production, levels of neutralizing antibodies, immunoglobulin A (IgA) and interferon-γ (IFN-γ), and histological changes of the intestine were analyzed in FRB-administered mice.

Results

FRB-administered immunocompetent mice without 5-fluorouracil (5-FU) treatment showed suppressed body weight loss caused by IFV-infection and rapid weight recovery. The virus yields in the lungs of FRB groups were reduced 3 days post-infection. Neutralizing antibody titers in the blood and respiratory tract and IgA levels in the lung and intestine were increased in FRB groups after 14 days of infection. An elevation in IFN-γ levels was observed in the blood of FRB-treated mice on days 3 and 14 after virus inoculation. In immunocompromised mice treated with 5-FU, oral FRB administration beginning 1 month prior to IFV inoculation resulted in significant suppression of body weight loss, reduced viral titers in the lung, elevated systemic and local neutralizing antibody titers, and preservation of normal colonic morphology compared with the control group. In contrast, when FRB was administered 1 h after viral inoculation, these protective effects were present but notably less pronounced than those observed with supplementation pre-inoculation.

Conclusions

FRB stimulates local and systemic adaptive immune functions, favors early recovery from influenza, and prolongs protective effects against IFV infection in both immunocompetent and immunocompromised mice.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12906-025-05240-y.

Background

Influenza is an acute, mild to severe respiratory infection caused by the influenza virus. According to the World Health Organization, seasonal influenza epidemics, caused by influenza A and B viruses, result in approximately one billion infections worldwide, with three to five million severe cases annually [1, 2]. Infections are particularly dangerous for high-risk immunosuppressed individuals, such as the elderly, pregnant women, and patients with transplanted organs, AIDS, or diabetes, as they are more likely to develop complications related to influenza infection [3, 4]. Therefore, optimal immune function should be maintained to prevent the development of influenza infection-related complications.

Antiviral agents are the main tools for controlling influenza. While neuraminidase inhibitors (oseltamivir and zanamivir) and cap-dependent endonuclease inhibitors (baloxavir marboxil) are frequently used drugs for treating influenza virus infections [5–7], drug-resistant viral strains continue to emerge [8, 9]. Although vaccines are indispensable tools for preventing influenza, their preparation based on embryonated chicken eggs takes time, and manufacturing of suitable vaccines against genetically mutated strains is challenging [10, 11]. To overcome these issues of vaccine production and therapeutic drug development, other types of agents that exert both inhibitory effects against the virus and different mechanisms of action, including stimulation of host immunological functions, are urgently needed.

Rice bran is a byproduct of rice milling and under-utilized as human food [12]. Bioactive components isolated from rice bran exhibit antioxidant activity [13, 14]. Additionally, these bioactive components exhibit antiviral properties. For example, rice bran reduces human rotavirus diarrhea via gut barrier function and innate immunity stimulation in animal models [15]. Fermentation increases the nutritional quality of rice bran and renders it suitable for human consumption. Fermented rice bran (FRB) is used as a dietary supplement [16] and exhibits a variety of biological activities, including anticancer activity [17, 18]. In addition, FRB alleviates colitis and prevents metabolic complications in mouse models [19]. Farmy et al. reported moderate anti-influenza virus activity of FRB in an in vitro assay system [20]. An aqueous extract obtained from fermented rice exhibited anti-influenza A virus (IFV) activity in vitro, possibly via inactivation of viral particles through viral membrane envelope disruption [21].

Recently, an FRB supplement (a unique probiotic product of rice bran fermented with a mixture of six microorganisms: Bacillus amyloliquefaciens M4, B. subtilis M5, B. sp. M6, Lactobacillus casei, Bifidobacterium bifidum, and Aspergillus oryzae) has been reported to counteract high-fat-induced obesity in mice by regulating gut microbiota and host metabolism [22]. However, the effect of this FRB supplement on IFV infection remains unknown.

Therefore, the present study was aimed at determining the effects of FRB on influenza in both immunocompetent and immunocompromised mice [treated with 5-fluorouracil (5-FU)].

Materials and methods

Materials

FRB was provided by MAXPROBIO Co., Ltd. (Shiga, Japan). FRB was produced by fermentation of rice bran using a mixture of six bacterial strains: B. amyloliquefaciens M4, B. subtilis M5, B. sp M6, L. casei (#NBRC 15883), B. bifidum (#NBRC 100015), and A. oryzae (#NBRC 6215). Rice bran (300 kg) was inoculated with a mixture of these bacterial strains (5 kg each) and maintained at < 50 °C in 0.06% glucose (w/w) solution, and the temperature was decreased gradually to < 30 °C 1 month later. FRB was dried until the moisture content reached < 12%, as previously described [22]. The cell numbers in the FRB were estimated to be 2.64 ± 1.09 × 10⁹ cells/g FRB [22]. FRB was ground in a mortar, passed through a sieve with a wire diameter of 200 µM, suspended in sterile water, and administered to mice orally using a feeding needle. The anti-influenza virus drug oseltamivir phosphate [(-)-ethyl (3R, 4R, 5 S)-4-acetamide-5-amino-3-(1-ethylpropoxy) cyclohex-1-ene-1-carboxylate monophosphate] was obtained from Hoffman-La Roche, Ltd. (Basel, Switzerland).

Cell and virus

Host cells, Madin-Darby canine kidney (MDCK) cells, were obtained from Denka Seiken Co., Ltd. (Tokyo, Japan) and grown in Eagle’s minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS) and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin; Nacalai Tesque, Inc., Kyoto, Japan). IFV (A/NWS/33, H1N1 subtype) was obtained from Denka Seiken Co., Ltd., and grown in MDCK cells. For plaque titration, MDCK cell monolayers were incubated in 35-mm dishes for 1 h at 37 °C with the virus, which was ten-fold serially diluted with phosphate-buffered saline (PBS). The cell monolayers were overlaid with MEM supplemented with 0.5% ME-agarose (Nacalai Tesque) and further incubated at 37 °C. Two days later, the cell cultures were fixed with a 10% formaldehyde solution and stained with a crystal violet solution for plaque counting.

Animals

Specific pathogen-free female BALB/c mice (5–6 weeks old) were purchased from Japan SLC (Shizuoka, Japan) and kept at a BSL-2 laboratory at the Center for Education in Laboratory Animal Research, Chubu University, under standard laboratory conditions (room temperature 22 ± 2 °C, relative humidity 50 ± 10%) and a 12/12-h light/dark cycle. Approval was obtained from the Animal Care Committee at Chubu University (permission number: 3010057), and all animal experiments were conducted in accordance with the animal experimentation guideline of Chubu University. A humane endpoint was applied: Animals that experienced more than 20% weight loss within 2 days were euthanized with sodium pentobarbital (150 mg/kg).

Animal experiments

We evaluated the effects of FRB based on body weight, viral replication, and virus-specific antibody [neutralizing antibody and immunoglobulin A (IgA)] and interferon-γ (IFN-γ) production in IFV-infected mice. After anesthesia by intraperitoneal injection of a mixture of Domitor (Orion Corporation, Espoo, Finland), midazolam (Sandoz, Tokyo, Japan), and Vetorphale (Meiji Seika Pharma, Tokyo, Japan; 0.75, 4, and 5 mg/kg, respectively) in 100 µL of sterile PBS, mice (five per treatment) were intranasally inoculated with IFV at 1 × 10⁴ plaque-forming units (PFU) in 50 µL of PBS on day 0. In the immunocompetent groups without 5-FU treatment, FRB (20 mg/day; 470–510 mg/kg/dose) suspended in sterile water was orally administered twice daily for 44 days (from 30 days before to 14 days after virus inoculation; Pre-1 M group) or 74 days (from 60 days before to 14 days after viral inoculation; Pre-2 M group). Oseltamivir phosphate (0.2 mg/day) dissolved in sterile water was orally administered twice daily for 14 days after viral inoculation. Mice in the control group were administered only sterile water (vehicle). Stool was collected from each mouse 30 and 60 days before virus inoculation and 0 and 14 days after inoculation. To determine fecal IgA levels, collected stool samples were disrupted with PBS supplemented with a protease inhibitor cocktail (Sigma Aldrich Japan, Tokyo, Japan) and vortexed. Stool sample homogenates were centrifuged at 815 ×g for 15 min, and the supernatant was collected as a fecal suspension. Bronchoalveolar lavage fluid (BALF) was collected from each mouse on days 3 and 14 post-inoculation by washing four times with 0.8 mL of PBS via a tracheal cannula and centrifugation at 815 ×g for 15 min. The supernatant was stored at -80 °C. Lung samples were collected on day 3 post-inoculation and sonicated for 10 s after addition of 10 µL of PBS per 1 mg of tissue followed by centrifugation at 9,060 ×g for 30 min. The supernatant was collected and stored at -80 °C. Virus yields on MDCK cells of the lung samples were measured using a plaque assay. Blood samples collected on days 3 and 14 post-inoculation were centrifuged at 815 ×g for 10 min, and the sera were stored at -20 °C. In the immunocompromised group (n = 7 mice per treatment), 5-FU was administered subcutaneously every other day at a dose of 0.25 mg/100 µL per injection (12.5 mg/kg), beginning 7 days prior to viral inoculation and continuing until 13 days post-inoculation. FRB (20 mg/day) was orally administered, either twice daily starting 1 month before viral inoculation or 1 h after inoculation. Blood, lung, BALF, macrophage, and stool samples were collected on days 3 and/or 14 post-inoculation for further analysis, as described below. The experimental dosing and treatment conditions are summarized in a timeline table in Fig. 1.

Fig. 1.

Timeline of animal experiments. Panel A is for immunocompetent groups, and B for immunocompromised groups

Assay for macrophage activity

Peritoneal macrophages were collected by washing the peritoneal cavity of mice with 0.8 mL of ice-cold PBS. The collected cells were suspended in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS. Cell suspensions (5 × 10⁵ cells/mL) were seeded into 24-well plates containing cover glasses and incubated for 1 h at 37 °C to allow cell adherence. Non-adherent cells were removed by washing with PBS, and fresh medium containing 0.025% fluorescent latex beads (diameter: 0.75 µM; Fluoresbrite YG Carboxylate Microspheres, Polysciences, Inc., Warrington, PA, USA) was added to each well. The cells were incubated for 4 h at 37 °C. Following incubation, the cells were washed three times with ice-cold PBS and fixed with 4% formaldehyde for 30 min. Macrophages that had phagocytosed fluorescent beads were identified and counted using fluorescence microscopy (OLYMPUS IX73, Olympus Corporation, Tokyo, Japan).

Assays for neutralizing antibody, IgA, and IFN-γ

Neutralizing anti-IFV antibody titers in the BALF and serum samples were determined using a 50% plaque reduction assay. Approximately 200 PFU of virus in 100 µL were mixed with 1–500-fold dilutions (100 µL) of BALFs or 20–50,000-fold dilutions (100 µL) of sera, and incubated at 37 °C for 1 h. MDCK cell monolayers in 35-mm dishes were infected with 100 µL of each mixture, incubated at 37 ˚C for 1 h, and overlaid with MEM supplemented with 0.5% ME-agarose. To determine the infectivity of the remaining virus, the plaques in each dish were fixed, stained, and quantified, as described above. The neutralizing antibody titer was defined as the highest sample dilution that reduced the plaque number by 50%, when compared to the PBS control. Mucosal IgA levels in BALFs and fecal suspensions were determined using an enzyme-linked immunosorbent assay (ELISA; Mouse IgA ELISA kit, Bethyl Laboratories, Inc., Montogomery, TX, USA). The IFN-γ concentration in serum samples was determined using a mouse IFN-γ kit (PGI Proteintech Group, Inc., Rosemont, IL, USA) according to the manufacturer’s instruction.

Histological analysis of the intestine

Jejunal and colonic tissues were collected from immunocompromised mice on day 14 post-infection (p.i.). The tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 2.25-µm-thick sections. Slides were prepared and tissues stained with hematoxylin and eosin (HE) to assess pathological changes. Stained sections were examined under a light microscope (OLYMPUS IX73, Olympus Co., Tokyo, Japan).

Statistics

Comparisons between two groups were performed using the Student’s t-test. Statistical significance was set at p < 0.05.

Results

Effects of FRB in immunocompetent mice infected with IFV

We aimed to compare the effects between animals pretreated with FRB for 1 and 2 months to confirm the optimal pretreatment timing and achieve sufficient effects in virus-loaded animals. Immunocompetent mice were infected with IFV and treated with FRB to evaluate the therapeutic effect of FRB under normal immune conditions. Clinical outcomes, virological parameters, and immune responses were subsequently assessed.

Bodyweight change of mice upon virus inoculation

No FRB side effects, such as diarrhea, were detected during the experimental period. Mice did not lose more than 20% of their body weight within 48 h of FRB administration. The weight of mice in the control group, administered with water, decreased by approximately 25% 1 week after virus inoculation; then, the mice gradually gained body weight until 14 days post-inoculation (Fig. 2). No marked weight loss was observed in oseltamivir-administered mice (by less than 5%) during the 14-day experimental period following virus inoculation, and the mice exhibited a significant body weight increase (p < 0.05, p < 0.01) 3–11 days p.i. Mice treated with 20 mg/day FRB for 1 (Pre-1 M) or 2 months (Pre-2 M) showed 10 and 13% weight loss 5 and 6 days p.i., respectively. Compared with control mice, FRB-administered mice showed no prolonged weight loss and exhibited rapid weight recovery. Both FRB-administered groups showed a significant body weight increase (p < 0.05, p < 0.01) between days 3 and 11 p.i.

Fig. 2.

Bodyweight changes in immunocompetent mice administered with fermented rice bran (FRB) and oseltamivir. Influenza A virus (IFV)-inoculated BALB/c mice (n = 5 per group) were administered distilled water, 0.2 mg/day oseltamivir, or 20 mg/day FRB 1 month (Pre-1 M) or 2 months (Pre-2 M) before virus inoculation. The bodyweight on the day of virus inoculation (day 0) was set to 100%. Data represent the mean ± standard deviation *p < 0.05, **p < 0.01 versus the control group

Effects of FRB on viral replication in virus-infected mice

The virus yields in lung samples obtained from IFV-inoculated BALB/c mice peaked 3–4 days post-inoculation and then decreased gradually until day 7 p.i. (data not shown). To clarify the effect of 20 mg/day FRB on IFV replication, viral titers in the lungs were determined 3 days p.i. (Fig. 3). Virus titers in both the Pre-1 M (p < 0.01) and Pre-2 M groups (p < 0.001) were significantly lower than those in the control group. No significant differences in viral titers were observed between the Pre-1 M and Pre-2 M groups. Oseltamivir significantly suppressed viral replication in the lungs (p < 0.001).

Fig. 3.

Virus yields in lung samples of immunocompetent mice after three days of Influenza A virus IFV inoculation. IFV-inoculated mice (n = 5 per group) were administered distilled water, 0.2 mg/day oseltamivir, or 20 mg/day FRB 1 month (Pre-1 M) or 2 months (Pre-2 M) before virus inoculation. Each value represents the mean ± standard deviation. **p < 0.01, ***p < 0.001 versus the control group

Effects of FRB on local and systemic virus-specific antibody responses in mice infected with virus

Neutralizing antibody titers were analyzed in BALF and serum samples obtained on days 3 and 14 after virus inoculation to clarify the effects of FRB on IFV-specific antibody production. The neutralizing antibody titers of all samples obtained 3 days p.i. were below the detection limit (data not shown). However, 14 days p.i., the neutralizing antibody titers in BALF (Fig. 4A) were significantly higher in FRB-treated mice (Pre-1 M and Pre-2 M; p < 0.05). Moreover, neutralizing antibody titers in the sera (Fig. 4B) obtained from the Pre-1 M and Pre-2 M groups were also significantly higher (p < 0.05 and p < 0.01, respectively) than those in sera obtained from control mice. No significant differences were observed between mice in the Pre-1 M and Pre-2 M groups. Antibody levels in both sera and BALF from the oseltamivir group were significantly lower (p < 0.01) than those in the control group (Fig. 4).

Fig. 4.

Neutralizing antibody titers in bronchoalveolar fluids (BALFs) and sera of immunocompetent mice obtained 14 days after virus inoculation. IFV-inoculated mice (n = 5 per group) were administered distilled water, 0.2 mg/day oseltamivir, or 20 mg/day FRB 1 month (Pre-1 M) or 2 months (Pre-2 M) before virus inoculation. Titers in BALF (A) and serum samples (B) are shown. Each value represents the mean ± standard deviation. *p < 0.05, **p < 0.01 versus the control group

Effects of FRB on IgA and IFN-γ production in virus-infected mice

The IgA levels in the respiratory organs (BALFs) and the intestine (feces) were analyzed using ELISA to evaluate whether FRB affects the secretion of IgA from the mucosa. Fourteen days after virus inoculation, the two FRB-treated groups produced significantly higher fecal IgA levels (p < 0.05) than the control group (Fig. 5A). IgA levels in BALF obtained 14 days p.i. from the Pre-1 M and Pre-2 M groups were significantly higher (p < 0.05 and p < 0.01, respectively) than those in the control group (Fig. 5B). The Pre-1 M and Pre-2 M groups showed similar IgA production tendencies in both BALF and fecal samples, and no significant differences were found between the two groups. In contrast, in the oseltamivir-treated group, IgA levels measured in the feces were comparable to those of the control group (Fig. 5A). In addition, IgA levels in BALF samples obtained from the oseltamivir-treated group were lower (p < 0.05) than those in the control group (Fig. 5B).

Fig. 5.

Mucosal IgA levels in feces and BALF samples of immunocompetent mice collected 30 days before (white bar) or 0 (gray bar) and 14 days (black bar) after virus inoculation. IFV-inoculated mice (n = 5 per group) were administered distilled water, 0.2 mg/day oseltamivir, or 20 mg/day FRB 1 month (Pre-1 M) or 2 months (Pre-2 M) before virus inoculation. The levels of IgA in feces (A) and BALF (B) were determined using an ELISA assay. *p < 0.05, **p < 0.01 versus the control group

We further determined the levels of IFN-γ in the sera of IFV-inoculated mice after administration of FRB using ELISA. The two groups treated with FRB 1 or 2 months before virus inoculation had significantly higher levels of IFN-γ (p < 0.01) in the blood than the control group both 3- and 14-days p.i. (Fig. 6).

Fig. 6.

Interferon (IFN)-γ levels in the sera of immunocompetent mice collected 3 and 14 days after virus inoculation. IFV-inoculated mice (n = 5 per group) were administered distilled water, 0.2 mg/day oseltamivir, or 20 mg/day FRB 1 month (Pre-1 M) or two months (Pre-2 M) before virus inoculation. The levels of IFN-γ at 3 (open column) or 14 days (closed column) post-inoculation were determined using an ELISA assay. ##p < 0.01, ###p < 0.001 versus the control group on day 3 post-inoculation. **p < 0.01, ***p < 0.001 versus the control group on day 14 post-inoculation

Effects of FRB in immunocompromised mice infected with IFV

Mortality, macrophage activity, and body-weight changes following viral inoculation

Mortality outcomes of 5-FU-treated mice are summarized in Table 1. The mortality rates in the control, Pre-1 M, and Post-1 H groups (FRB treatment initiated 1 h after inoculation) were 14, 0, and 14%, respectively.

Table 1.

Effects of FRB on the mortality and macrophage activity of influenza virus-infected mice treated with 5-fluorouracil

Group	Mortarlity		Macrophage activity
	Survivors/total (%)	Day of death	(% of cells incorporating beads)
Control	6/7 (14)	8 days p.i.	16.7 ± 2.4
FRB (Pre-1 M)	7/7 (0)	–	26.6 ± 3.6 ***
FRB (Post-1 H)	6/7 (14)	9 days p.i.	21.9 ± 2.6**

Macrophage activity was determined at 14 days post-infection (p.i.)

**p < 0.01, ***p < 0.001 vs. control

Macrophage activity, assessed on day 14 p.i, demonstrated enhanced phagocytic function in FRB-treated groups. Both the Pre-1 M and Post-1 H groups exhibited significantly higher macrophage phagocytosis than the control group (p < 0.001, p < 0.01, respectively), as shown in Table 1.

Body weight changes in immunocompromised mice were more prolonged and severe than those in immunocompetent mice, with weight loss persisting until 8 days p.i. (Fig. 7 vs. Figure 2). FRB treatment significantly attenuated weight loss between days 3 and 14 p.i. and promoted more rapid recovery compared with the control group. While the Pre-1 M group showed higher suppression of weight loss than the Post-1 H group, the difference between the two groups was not significant.

Fig. 7.

Bodyweight changes in immunocompromised mice administered FRB. IFV-inoculated immunocompromised mice treated with 5-fluorouracil (5-FU; 12.5 mg/kg; n = 7 per group) were orally administered distilled water (control) or 20 mg/day FRB starting 1 month before (Pre-1 M) or 1 h after virus inoculation (Post-1 H). Bodyweight on the day of virus inoculation (day 0) was set to 100%. Data are presented as the mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group

Effects of FRB on viral replication in the lungs of virus-infected mice

The impact of FRB treatment on viral replication in the lungs was assessed by measuring lung virus titers on day 3 p.i. Mice in the Pre-1 M group exhibited a significant (51%) reduction in lung virus titers compared with those in the control group (p < 0.01; Fig. 8). Although the Post-1 H group also showed an 19% reduction in viral titers compared with the control group, this difference was not significant. The viral titer in the Pre-1 M group was significantly lower than that in the Post-1 H group (p < 0.01). Given that prolonged IFV replication has been reported in immunocompromised hosts, such as transplant recipients [23, 24], viral presence in lung tissue was also evaluated on day 14 p.i.; however, no virus was detectable in any of the lung samples from the control, Pre-1 M, or Post-1 H groups (data not shown), indicating complete viral clearance by this time point.

Fig. 8.

Virus titers in lung tissues of immunocompromised mice on day 3 post-infection. IFV-inoculated mice with 5-FU treatment (n = 7 per group) were administered distilled water (control) or 20 mg/day FRB starting 1 month before (Pre-1 M) or 1 h after virus inoculation (Post-1 H). Each value represents the mean ± standard deviation. ***p < 0.001 versus the control group. ^##p < 0.01 versus Post-1 H group

Effects of FRB on systemic and local neutralizing antibody responses in virus-infected mice

Neutralizing antibody titers were measured in serum, BALF, and feces following influenza virus infection to evaluate the impact of FRB on humoral immunity in immunocompromised mice. In serum samples collected on day 3 p.i., the Pre-1 M group exhibited significantly higher neutralizing antibody titers, which were approximately 2.5- and 2-fold greater than those in the control and Post-1 H groups, respectively (p < 0.001; Fig. 9A). No significant difference was observed between the control and Post-1 H groups at this early time point. By day 14 p.i., serum antibody titers increased markedly in all three groups (Fig. 9B). However, titers remained significantly higher in both the Pre-1 M and Post-1 H groups relative to the control group (p < 0.001 for Pre-1 M; p < 0.01 for Post-1 H). Additionally, a significant difference in serum antibody titers was observed between the Pre-1 M and Post-1 H groups on day 14 p.i. (p < 0.01), indicating a more robust systemic response with earlier FRB administration.

Fig. 9.

Neutralizing antibody titers in sera of immunocompromised mice obtained 3 (A) and 14 days (B) after viral inoculation. IFV-inoculated mice with 5-FU treatment (n = 7 per group) were administered distilled water (control) or 20 mg/day FRB starting 1 month before (Pre-1 M) or 1 h after virus inoculation (Post-1 H). Each value represents the mean ± standard deviation. **p < 0.01, ***p < 0.001 versus the control group. ^##p < 0.01, ^###p < 0.001 versus Post-1 H group

Neutralizing antibody titers in BALF and fecal samples were measured on day 14 p.i. to assess the effect of FRB on local mucosal immunity. In BALF, titers were significantly elevated in both the Pre-1 M and Post-1 H groups, approximately 1.7- and 1.5-fold, respectively, compared with those in the control group (p < 0.001; Fig. 10A). Similarly, fecal samples from the Pre-1 M and Post-1 H groups showed 3.5- and 3.2-fold higher neutralizing antibody titers, respectively, than controls (p < 0.01; Fig. 10B). No significant differences were observed between the Pre-1 M and Post-1 H groups for either BALF or fecal antibody titers.

Fig. 10.

Neutralizing antibody titers in bronchoalveolar lavage fluid (A) and fecal (B) samples of immunocompromised mice obtained 14 days after virus inoculation. IFV-inoculated mice with 5-FU treatment (n = 7 per group) were administered distilled water (control) or 20 mg/day FRB starting 1 month before (Pre-1 M) or 1 h after virus inoculation (Post-1 H). **p < 0.01, ***p < 0.001 versus the control group

Effects of FRB on morphological changes in the intestine of virus-infected mice

The impact of FRB on intestinal morphology in IFV-infected, 5-FU-treated immunocompromised mice was assessed at 14 days p.i. using histological analysis. HE-stained sections of jejunal tissues from both control and FRB-treated mice showed no marked changes compared with uninfected, 5-FU-untreated mice (Fig. 11A). However, in the colon, marked tissue thinning was observed in the control and Post-1 H groups following IFV infection, relative to uninfected, 5-FU-untreated normal mice (Fig. 11B, C). Colon thickness was measured using a caliper. Mice in the control and Post-1 H groups had significantly lower colon thickness [0.89 ± 0.096 mm (p < 0.001) and 0.91 ± 0.080 mm (p < 0.001), respectively] than mice in the Pre-1 M group (1.56 ± 0.11 mm) (Table S1). No histological abnormalities other than thinning were observed in the control and Post-1 H groups compared with the Pre-1 M group. Colonic tissue from the Pre-1 M group exhibited no apparent histological abnormalities and maintained structural integrity comparable to that of normal, uninfected mice.

Fig. 11.

Histological and gross anatomical assessment of intestinal tissues in virus-infected, immunocompromised mice on day 14 post-infection. Influenza virus-inoculated mice with 5-FU treatment were administered distilled water (control) or 20 mg/day FRB starting 1 month before (Pre-1 M) or 1 h after virus inoculation (Post-1 H). Representative sections of the jejunum (A) and colon (B) were stained with hematoxylin and eosin (scale, 200 µM). Photographs of the colon are shown for comparison of the thickness of the colon between the groups (C). Red arrows indicate a thinner colon

Discussion

Mice are commonly used species to study infections caused by influenza virus [25, 26]. In this study, we aimed at determining the effects of FRB on influenza in immunocompetent or immunocompromised mice infected with IFV. Anorexia and dehydration are common clinical signs of influenza and lead to excessive body weight loss in mice. In the present study, BALB/c mice without 5-FU treatment in the control group began to lose body weight approximately 4 days after inoculation with IFV (A/NWS/33 strain), with peak weight loss occurring approximately 7 days p.i. (Fig. 2). FRB has been shown to reduce the percentage of body weight loss, which leads to a quicker recovery from infection. Since influenza infection induces a sex-specific immunological response, and host sex influences viral replication [27–29], it might be important to compare viral replication between male and female BALB/c mice.

Respiratory infections pose a global health threat in both developed and developing countries [30]. Viruses are major pathogens of these infections, being responsible for more than 90% of lower respiratory infections [31]. The only way to prevent the spread of infections is to improve the immune function of the host [32]. Influenza is a respiratory tract infection that affects the host immune response to pathogens. When viruses invade a host, they elicit an immune response, which can be subdivided into innate immunity and adaptive immunity. Natural killer cells are innate lymphocytes that function as immune regulators before the development of pathogen-specific immune responses [33–36]. These cells are activated following IFV infection, and their main functions are IFN-γ production and killing of infected cells [33–35, 37]. In the present study, increased IFN-γ levels were observed in the blood of FRB-administered mice (Fig. 6), which may play a role, at least in part, in suppressing viral replication in the lung (Fig. 3).

Neutralizing antibodies are a major factor in adaptive immunity and responsible for protection against challenge with influenza viruses of the same strain. Neutralizing antibodies, including IgA, which is secreted from the mucosa and plays a crucial role in the progression of influenza [38], inhibit the entry of viral particles and can also inhibit the conformational changes necessary to expose the fusion peptide on hemagglutinin (HA), which is a glycoprotein present in the envelope of the virion [39]. Antibodies against IFV are produced following natural infection or vaccination. Vaccines are the best countermeasure against influenza; however, the immune responses induced by influenza vaccines, such as whole inactivated virus, split virus, and recombinant HA-based vaccines, are short-lived, and their effectiveness can decline even within one season. In contrast, natural influenza virus infection can induce longer-lived immune responses [40]. As oral FRB administration elevated neutralizing and mucosal antibody titers (Figs. 4 and 5), it might be worth assessing whether the duration of the immune responses induced by vaccination could be extended by FRB intake.

Bacteria are known to enter M cells in Peyer’s patches [41, 42], where they are subsequently phagocytosed by antigen-presenting cells, including macrophages and dendritic cells [43, 44]. This contributes to immune responses such as the production of cytokines [45]. If these phenomena occur in the intestine of mice administered with FRB, which contains bacteria, some bacteria could reach Peyer’s patches and enter M cells, thus triggering immune responses. These functions of Peyer’s patches may underlie the observed production of IFN-γ and virus-specific antibodies in FRB-administered mice.

In the present study, we used oseltamivir phosphate, an influenza medication, as the positive control. The orally administered drug markedly inhibited IFV growth and simultaneously suppressed IFV-specific antibody production in IFV-infected mice (Figs. 3 and 4). In contrast, FRB suppressed viral replication and stimulated antibody production (Figs. 3 and 4). Synthetic antiviral agents, such as oseltamivir, are associated with several limitations, including severe adverse reactions and drug resistance [46]. Therefore, alternatives that are more potent, safer, and can overcome drug resistance are needed. In addition, because the activity of FRB against influenza virus is not dependent on virus-specific enzymes but mainly on the immunological functions of the host, drug resistance may not occur by FRB intake.

The FRB used in this study contained proteins, lipids, and carbohydrates as macronutrients along with fibers, minerals, and vitamins [22]. Microbial analyses revealed that the abundance of the three major bacterial genera, Bacillus, Pediococcus, and Staphylococcus spp., in the FRB was 45.7, 21.6, and 17.5%, respectively, and administration of FRB to mice caused considerable alterations in the gut microbiota [22]. Such modulation of the gut microbiota could improve the host immune function and improve the levels of secretory IgA and neutralizing antibodies, as observed in IFV-infected mice (Figs. 4 and 5).

Our previous study demonstrated that subcutaneous injection of 5-FU in mice serves as an effective model of immunosuppression [47]. In the present study, FRB enhanced the phagocytic activity of intraperitoneal macrophages obtained from 5-FU-treated mice (Table 1). Phagocytosis of foreign substances, including pathogens such as influenza virus, initiates the innate immune response and subsequently activates the adaptive immune response [48–51]. Thus, it is reasonable that FRB may contribute to virus-specific antibody production, at least in part, through its stimulation of macrophage-mediated phagocytosis.

Histological examination revealed that IFV-infected, 5-FU-treated mice exhibited substantial thinning of the colonic wall on day 14 p.i. (Fig. 11). This morphological alteration was prevented in the Pre-1 M group, in which FRB was administered orally starting 1 month before viral inoculation. However, no such protective effect was observed in the Post-1 H group, when FRB administration commenced 1 h p.i. These findings suggest that prevention of IFV-induced colonic damage in immunocompromised mice requires pre-exposure FRB supplementation.

In the absence of FRB-administration, IFV-infected control mice exhibited statistically significant narrowing of the colon (Fig. 11B, C). Previous studies have demonstrated anti-colitic effects of FRB in mice subjected to dextran sodium sulfate-induced colitis, showing preservation of colon length [52] and reduced intestinal damage [53]. Notably, previous studies did not report narrowing of the colon, as observed in the present study. Thus, FRB does not appear to be associated with narrowing of the colon. It is well established that influenza virus infection in mice can cause damage to the respiratory and intestinal tracts [54–57]. In humans, influenza virus infection of the respiratory tract can cause both respiratory and non-respiratory symptoms. Gastrointestinal symptoms such as anorexia, nausea, vomiting, diarrhea, and abdominal pain can occur in individuals with influenza infection. However, it has not been directly demonstrated that influenza virus replicates in the gastrointestinal tract, despite virus mRNA and virus detection in human intestinal tissues and stool [58–60]. Thus, the extent to which influenza virus can infect and replicate in gastrointestinal tissues remains understudied. While changes in the intestinal microbiota following respiratory IFV infection have been reported, direct viral infection of intestinal tissues has not been confirmed [56, 58]. Histological analyses of IFV-infected mice have revealed inflammatory cell infiltration in the colon, but no pronounced narrowing of the colon [54, 57]. 5-FU, a chemotherapeutic agent used to treat colorectal cancer, causes gastrointestinal side effects, including intestinal injury [61–65]. In previous studies, 5-FU at doses ranging from 40 to 450 mg/kg, 3.2- to 36-fold higher than the 12.5-mg/kg dose used in the present study, induced intestinal damage and inflammation [62–65]. However, no colonic narrowing was reported in those studies. Therefore, the combination of IFV infection and low-dose 5-FU treatment may underlie the unique colonic narrowing observed in the present study (Fig. 11).

As a probiotic product, FRB might offer additional benefits through modulation of intestinal microbiota and its metabolites, potentially improving influenza outcomes by attenuating lung inflammation and reducing viral load [66–69].

Conclusions

Although antiviral agents and vaccines are available, influenza virus infection remains a serious health problem. Therefore, it is important to identify alternative, safe, and cost-effective therapeutic and prevention agents against infectious respiratory diseases caused by influenza virus. In the present study, we evaluated the effects of FRB prepared using a mixture of six strains of microbes on influenza by evaluating bodyweight changes and virus yields in the lungs of both immunocompetent and immunocompromised mice infected with influenza virus. We further evaluated the preventive effects of FRB on influenza virus infection by estimating the levels of neutralizing antibodies, secretory IgA, and cytokines in a mouse model. Daily consumption of probiotic-based fermented products, including FRB, could improve the host’s health status, especially its immunological status, and aid in resisting respiratory infections. Further studies should elucidate the mechanisms underlying the immunological effects of FRB, e.g., by analyzing the villi in the intestine, immune cell populations in Peyer’s patches and the spleen, and gene expression in Peyer’s patches via DNA microarray analysis.

Abbreviations

BALF

Bronchoalveolar lavage fluid

ELISA

Enzyme-linked immunosorbent assay

FBS

Fetal bovine serum

FRB

Fermented rice bran

HA

Hemagglutinin

HE

Hematoxylin and eosin

IFV

Influenza A virus

IgA

Immunoglobulin A

IFN-γ

Interferon-γ

MDCK

Mardin-Darby canine kidney

MEM

Eagle’s minimum essential medium

PBS

Phosphate-buffered saline

PFU

Plaque-forming unit

p.i.

Post-infection

5-FU

5-Fluorouracil

Authors’ contributions

Design of the experiments, KH, SA, YM, KM, and TK; preparation of materials, YM, KM, and TW; investigation, KH, SA, YM, and MH; analysis of the data, KH, SA, JBL, and TK; writing–original draft preparation, KH, and SA; writing–review and editing, KH JBL, SA, and TK. All authors have read the manuscript.

Funding

Part of this work was supported by JST-Mirai, Grant Number JPMJMI22D2. The work was also supported by a Grand-in-Aid for Scientific Research (C; No. 22K04902) by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. Part of this research was also supported by the Daiko Foundation, and the Cooperative Research Program of “Network Joint Research Center for Materials and Devices.

Data availability

Data are provided within the manuscript. Any additional data and materials will be provided upon request from the corresponding author.

Declarations

Ethics approval and consent to participate

The animal study protocol was approved by the Institutional Review Board of Chubu University (protocol code 3010057 and date of approval April 20, 2020).

Consent for publication

Not applicable.

Competing interests

Yoshiteru Maehara and Kazuo Maehara are employees of MAXPROBIO Co., Ltd.