2022 Volume 28 Issue 4 Pages 335-341
Lactobacillus paracasei K71 (K71) isolated from Japanese sake lees (Sakekasu) was previously shown to enhance IL-12 production in mouse spleen cell culture. In this study, we examined whether heat-killed K71 exerts immunostimulatory effects in normal BALB/c mice. Six-week dietary supplementation with 0.2% heat-killed K71 resulted in significantly higher levels of serum IgG2a and fecal IgA than control mice. Upon administration, heat-killed K71 increased IFN-γ production in Peyer's patch cells while simultaneously suppressing IL-4 production in spleen cells. Spleen cells from mice fed heat-killed K71 exhibited a significant increase in cytotoxicity against YAC-1 cells. Furthermore, splenic CD49b+NKp46+ NK cells from mice fed heat-killed K71 showed significantly enhanced IFN-γ production. These results suggest that oral intake of K71 may augment Th1 type immune function and innate immunity, thereby potentially preventing infections and/or tumorigenesis.
Lactic acid bacteria (LAB) are fundamental microorganisms that have been widely used in manufacturing fermented foods and have received increasing attention for their health promoting properties. Gut commensal LAB strains of human origin have been used as probiotics for disease prevention and health promotion (Fontana et al., 2013). Food-derived LAB strains, especially those from fermented foods, have also been found to exert immunomodulatory effects and improve intestinal function (Zielinska and Kolozyn-Krajewska, 2018). However, not all LAB strains have been shown to possess health promoting properties and provide health benefits to the host (Azais-Braesco et al., 2010). Additionally, the health promoting properties of LAB vary considerably among strains.
The immunomodulatory effects of LAB are thought to be due to the bacterial components and products, which have been demonstrated to be effective even when consumed as dead bacteria (Pique et al., 2019). LAB cell components, including lipopolysaccharides, lipoteichoic acids and unmethylated cytosine and guanine (CpG)-DNA, affect the ability of immune cells to produce cytokines, which is critical for the immunomodulatory effects of LAB (Taverniti and Guglielmetti, 2011). It is assumed that the cytokine production pattern triggered by each LAB strain is different and, accordingly, the effect on immune function is different (Barberi et al., 2015; Ding et al., 2017; Dong et al., 2012).
The adaptive immune system, which produces responses highly specific to particular antigens, consists of cellular immunity and humoral immunity, which are stimulated by distinct cytokines produced by helper T cell type 1 (Th1) and Th2 cells, respectively. Th1 cells produce interferon (INF)-γ and interleukin (IL)-2, and promote cellular immune responses against intracellular viral and bacterial infection. Besides exogenous pathogens, cancer cells are eliminated by cytotoxic T cells, the primary effectors of cellular immunity, which is supported by Th1 cytokines such as IFN-γ. Th2 cells produce IL-4, IL-5 and IL-13, and promote humoral immune responses targeting extracellular pathogens, in which immunoglobulin is the primary component. Meanwhile, Th1 and Th2 cytokines maintain a relative balance, contributing to the maintenance of immune equilibrium. Under certain circumstances, the relative suppression of Th2 cells by the relative increase of Th1 activities, or vice versa, may be a critical mechanism for maintaining or restoring immune function.
Lactobacillus paracasei K71 (K71) isolated from Japanese sake lees (Sakekasu) was shown to enhance both IL-12 and INF-γ production and suppress IL-4 production in mouse spleen cell culture (Kumagai et al., 2013). In addition, oral intake of heat-killed K71 resulted in significant decreases in serum levels of total and ovalbumin (OVA)-specific IgE in mice sensitized with OVA in combination with alum adjuvant (Kumagai et al., 2013). These effects can be attributed to the suppression of Th2 responses via the induction of Th1 responses by K71 bacterial components; however, it is unconfirmed whether K71 intake leads to an enhanced Th1 response and/or Th1-dominant state in vivo. LAB that exert stimulatory activity in augmenting Th1 responses are expected to prevent bacterial and viral infections and to have anticancer effects by enhancing cellular immunity. Certain LAB strains belonging to Lactobacillus genera have been reported to protect mice against influenza virus infection with concomitant increases in Th1 type immune responses (Chiba et al., 2013; Kawashima et al., 2011; Matsusaki et al., 2016), natural killer (NK) cell cytotoxicity (Goto et al., 2013; Iwabuchi et al., 2012; Kawase et al., 2012; Nagai et al., 2011; Takeda et al., 2011; Yasui et al., 2004) and mucosal IgA production (Asama et al., 2017; Kawashima et al., 2011; Kikuchi et al., 2014). In this study, we investigated whether heat-killed K71 could enhance Th1 type immune responses as well as NK cell activity and IgA production in normal mice.
Animals and diets Six-week-old female BALB/c mice were purchased from Charles River Japan (Yokohama, Japan) and maintained conventionally at 23 ± 2 °C under a 12-h light-dark cycle. The dry powder form of heat-killed L. paracasei K71 (K71) was obtained from Kameda Seika Co., Ltd. (Niigata, Japan). Cultured K71 was heated at 120 °C for 10 s with a plate heater, and dried with a spray dryer. The absence of viable bacteria was confirmed using BCP plate count agar (Eiken Chemical, Tokyo, Japan). The animals were fed a standard diet (MF; Oriental Yeast, Tokyo, Japan) supplemented with or without 0.2% heat-killed K71 for 42 consecutive days (6 weeks). The daily dose of heat-killed K71 was estimated to be approximately 8 to 10 mg/day assuming that a mouse eats 4 to 5 g/day. Mice were treated in accordance with the guidelines for animal experiments as laid out by Niigata University. All experimental procedures were approved by The Ethics Committee for Animal Experiments of Niigata University (approval number 39).
Antibody quantification The concentrations of total IgG1 and IgG2a in serum and total IgA in fecal extracts were measured with ELISA using IgG1, IgG2a and IgA EIA kits (Bethyl Laboratories, Montgomery, TX, USA). To obtain serum, blood samples were collected from the tail vein of mice. Blood samples were allowed to clot for 30 min at room temperature, and were then centrifuged for 15 min at 1 000 × g to remove the clot and blood cells. For determination of IgA in feces, fecal extracts were prepared. Specifically, fecal pellets (100 mg) were suspended into 1 mL of phosphate-buffered saline (PBS) containing 0.1% sodium azide followed by extraction of IgA by vortexing for 5 min. Then, the samples were centrifuged at 10 000 × g for 5 min, and the supernatants were collected as fecal extracts for ELISA.
Cytokine production assay Mice were sacrificed and their spleens and Peyer's patches were obtained on day 42. Single cell suspensions of both spleen and Peyer's patch cells were prepared by crushing and pressing the respective organs through a 70 µm nylon mesh cell strainer (BD Falcon, Bedford, MA, USA) and removing red blood cells with lysis buffer, followed by washing twice with PBS. Both cell types were suspended at 2 × 106 cells/mL in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% (vol/vol) heat-inactivated fetal bovine serum (FBS; Roche, Mannheim, Germany), 100 U/mL penicillin and 100 µg/mL streptomycin and, using 96-well plates (Nunc, Roskilde, Denmark), stimulated with anti-CD3 (clone: 145-2C11) and anti-CD28 (clone: 37.51) antibodies (1 µg/mL each) for 72 h. After stimulation, culture supernatants were collected for measurement of IFN-γ and IL-4 using ELISA. The concentrations of IFN-γ and IL-4 were measured using the respective kits of ELISA MAX Standard Set (Biolegend, San Diego, CA, USA).
Cytotoxicity assay Splenic NK cell cytotoxicity was assessed by lactate dehydrogenase (LDH) release from NK cell-sensitive target cells, YAC-1. YAC-1 cells were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). Spleen cells (2×105 cells/well) (effector) were incubated with YAC-1 cells (target) at an effector to target ratio of 20:1 in 96-well U-bottom plates. After 6 h incubation, LDH released from damaged YAC-1 cells into the cell culture media was measured using a cytotoxicity detection kit (Roche, Mannheim, Germany), according to the manufacturer's instructions. NK cell cytotoxicity was calculated according to the formula: Cytotoxicity (%) = (Experimental value − Effector Cells Spontaneous Control value − Target Cell Spontaneous Control value) / (Target Cell Maximum Control value − Target Cell Spontaneous Control value) × 100.
Flow cytometric analysis of intracellular IFN-γ production by NK cells Intracellular IFN-γ production in NK cells, defined as CD3-CD49b+NKp46+ cells (Walzer et al., 2007), was assessed by flow cytometry. To induce cytokine production, spleen cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin in RPMI-1640 medium supplemented with 10% FBS containing 10 µg/mL brefeldin A, a protein transport inhibitor at 37 °C in a 5% CO2 humidified atmosphere for 4 h. After stimulation, cells were stained with anti-mouse CD3-PE (clone: 145-2C11), anti-mouse CD49b-PerCP (clone: DX5) and anti-mouse NKp46-APC (clone: 29A1.4) antibodies (all from Biolegend) and fixed with 4% paraformaldehyde in PBS, followed by permeabilization with Intracellular Staining Perm Wash Buffer (Biolegend). Intracellular cytokine staining was performed with anti-mouse IFN-γ-FITC (clone: XMG1.2) antibody (Biolegend) or isotype control antibody (Biolegend) in Intracellular Staining Perm Wash Buffer for 20 min in the dark at room temperature. Each of the stained cells was analyzed with a BD FACSCalibur instrument (BD Biosciences, San Jose, CA, USA), and IFN-γ levels were assessed as the mean fluorescence intensity (MFI) of approximately 1 000 cells using the Cell Quest software (BD Biosciences).
Statistical analysis Data are presented as mean ± standard error of the mean (SEM). Statistical differences between two groups were determined using Welch's t-test in Microsoft Excel (Version 2013, Microsoft, Redmond, WA, USA). Statistical significance was set at p < 0.05.
There is a growing body of experimental evidence that LAB can potentially affect immune function. LAB are the most representative probiotics, which are defined as viable microorganisms able to confer health benefits to the host. However, non-viable LAB have been shown to exert immunomodulatory effects, such as a predominant Th1 profile (Adams, 2010; Matsusaki et al., 2016), NK cell activation (Adams, 2010; Goto et al., 2013; Iwabuchi et al., 2012; Kawase et al., 2012; Takeda et al., 2011) and induction of enhanced mucosal IgA production (Adams, 2010; Asama et al., 2017; Kikuchi et al., 2014). Various LAB components such as lipoteichoic acids, peptidoglycans, lipopolysaccharides and DNA have immunomodulatory effects, likely through interaction with pattern recognition receptors (PRRs) on epithelial cells and immune cells, including dendritic cells, macrophages and lymphocytes (Plaza-Diaz et al., 2019). It is increasingly accepted that cell components derived from LAB are critical for their immunomodulatory effects, and that these effects are not necessarily dependent on the bacteria being alive (Adams, 2010; Plaza-Diaz et al., 2019). The use of non-viable LAB exhibiting probiotic properties confer advantages in reducing the risk that live bacteria could cause some pathology of their own and in manufacturing food products that are easier to store and have a long shelf-life (Adams, 2010). In the present study, we examined whether oral intake of heat-killed K71, which promotes IL-12 production in spleen cell culture, can induce Th1 responses, enhance NK activity and increase immunoglobulin levels in normal BALB/c mice.
The profile of immunoglobulin isotypes is influenced by the balance of Th1 and Th2 immune responses. Particularly in mice, IFN-γ and IL-4 induce IgG2a and IgG1 production, respectively (Snapper and Paul, 1987). Therefore, levels of IgG2a and IgG1 indirectly reflect Th1/Th2 immune responses. Six-week (42-d) dietary supplementation with 0.2% heat-killed K71 resulted in a significant increase in serum total IgG2a level and no significant difference in serum total IgG1 level (Fig. 1A, B), suggesting that K71 augments Th1 immune responses in mice. While IgG1 is the largest IgG subclass, IgG2a is the most potent IgG subclass in binding to FcγRI on monocytes, macrophages and dendritic cells in mice. FcγRI preferentially interacts with IgG2a and contributes substantially to protection against bacterial infection (Ioan-Facsinay et al., 2002). Mouse IgG2a also binds to FcγRIV on macrophages, which potentially contributes to antibody-dependent cell-mediated cytotoxicity (Bruhns, 2012). Increased IgG2a levels by K71 may contribute to the enhancement of host defenses against infection. Mice fed heat-killed K71 for 6 weeks also showed a significantly higher level of fecal IgA than control mice (Fig. 1C), suggesting that K71 cell components can promote intestinal IgA production. As is well-known, mucosal IgA neutralizes harmful bacteria and viruses, which inhibits their adherence to epithelial cells (Lamm, 1997). However, the relevance of IgA production to Th1/Th2 responses is currently less clear. K71 cell components likely have the ability to induce cytokines involved in the induction of IgA class switching and production in the intestine, including TGF-β, IL-21, IL-6, APRIL and BAFF (Tezuka and Ohteki, 2019).
Effects of heat-killed K71 on serum and fecal antibody levels. BALB/c mice were fed with a standard diet with or without 0.2% heat-killed K71 powder. After 42 d of feeding, the concentrations of total IgG1 (A) and IgG2a (B) in sera and total IgA (C) in fecal extracts were determined by ELISA. Data are expressed as mean ± SEM (n = 8) and were analyzed by unpaired two-tailed t-test with Welch's correction, *p < 0.05 vs control.
Next, we examined the ability of Peyer's patch cells and spleen cells to produce IFN-γ and IL-4, the principal cytokines of Th1 and Th2 cells respectively. Peyer's patch cells from mice fed heat-killed K71 for 6 weeks showed significantly increased IFN-γ production and no significant change in IL-4 production upon stimulation with anti-CD3 and anti-CD28 antibodies (Fig. 2A). The substantial increase in IFN-γ production provides direct evidence that heat-killed K71 can enhance Th1 responses in vivo. Bacterial and food antigens are taken up by Peyer's patches, a part of the gut-associated lymphoid tissue in the small intestine, which are responsible for inducing immune responses against foreign agents and commensal bacteria (Corr et al., 2008). Orally ingested K71 is quite likely to be incorporated into Peyer's patches, where K71 cell components may act on dendritic cells to induce Th1 cells. Meanwhile, spleen cells from mice fed heat-killed K71 showed no significant change in IFN-γ production and a significant decrease in IL-4 production (Fig. 2B). The production pattern of IFN-γ and IL-4 in spleen cells was not exactly identical to that in Peyer's patch cells. On the basis of the Th1/Th2 paradigm, our data obtained from spleen cells imply that heat-killed K71 causes a relative shift in the Th1/Th2 balance toward Th1 dominance due to decreased Th2. However, it is unclear how ingested K71 suppressed the production of IL-4 in spleen cells while simultaneously enhancing the production of IFN-γ in Peyer's patch cells. This discrepancy may have implications for understanding the immunomodulatory effects of K71 and is an issue for further study.
Effects of heat-killed K71 on cytokine production in Peyer's patch and spleen cells. Following dietary supplementation with 0.2% of heat-killed K71 for 42 d, Peyer's patche cells and spleen cells from mice were stimulated with anti-CD3 and anti-CD28 antibodies for 72 h, and the concentrations of IFN-γ (A) and IL-4 (B) in the supernatants were determined by ELISA. Data are expressed as mean ± SEM (n = 8) and were analyzed by unpaired two -tailed t-test with Welch's correction, *p < 0.05 vs control.
Certain strains of Lactobacillus have been shown to increase the activity of NK cells, which play a critical role in early defenses against cancer and infections (Ashraf and Shah, 2014). In this study, we investigated whether heat-killed K71 intake affects NK cell activity. A cytotoxicity assay against YAC-1 cells demonstrated that spleen cells from mice fed heat-killed K71 exhibited significantly increased cytotoxic activity (Fig. 3A). Besides their cytotoxicity for elimination of virus-infected cells and cancer cells, NK cells also play an important role as a source of IFN-γ production (Biron et al., 1999). To examine the ability of NK cells to produce IFN-γ, spleen cells were stimulated with PMA and ionomycin, and then CD49b+NKp46+ cells producing IFN-γ were detected using flow cytometry. As a result, significantly increased levels of IFN-γ were detected in CD49b+NKp46+ cells from mice fed heat-killed K71 compared to those from control mice (Fig. 3B), while no significant differences in the percentage of CD49b+NKp46+ cells were detected between the two groups. K71 was found to have the potential to enhance NK function, while the exact mechanism remains to be elucidated. Certain LAB have been shown to act on dendritic cells, thereby activating NK cells and enhancing IFN-γ production and cytotoxicity (Aziz and Bonavida, 2016). Cytokines, such as IL-12, IL-15 and IL-18, are known to be pivotal for NK cell development and activation (Walzer et al., 2005). Heat-killed K71 has previously been shown to significantly promote the production of IL-12 to activate NK cells in spleen cell culture (Kumagai et al., 2013). A plausible explanation for the observed increase in NK cell function is that orally ingested K71 induces cytokines, such as IL-12, IL-15 and IL-18, in dendritic cells, which in turn activate NK cells.
Effects of heat-killed K71 on NK cell function. Following dietary supplementation with 0.2% of heat-killed K71 for 42 d, cytotoxic activity of mouse spleen cells against YAC-1 cells was measured as NK cell cytotoxicity using LDH assay (A) and the ability of NK cells to produce IFN-γ was assessed by flow cytometry (B). To evaluate the cytotoxicity, spleen cells (effector) from each mouse were incubated with YAC-1 cells (target) at an effector:target ratio of 20:1 for 6 h. To evaluate the ability of NK cells to produce IFN-γ, spleen cells were stimulated with 50 ng/ml PMA and 1 µg/ml ionomicin for 4 h in the presence of 10 µg/ml brefeldin A. Intracellular IFN-γ levels of NK cells defined as CD3-CD49b+NKp46+ cells were assessed as the mean fluorescence intensity (MFI) of approximately 1 000 cells. Representative flow cytometric histograms and MFI values are shown (B right). Data are expressed as mean ± SEM (n = 8) and were analyzed by unpaired two-tailed t-test with Welch's correction, *p < 0.05 vs control.
In conclusion, our results showed the potential ability of heat-killed K71 to enhance Th1 responses, NK cell activity and intestinal IgA production, raising the possibility that oral intake of K71 may contribute to prevention of infections and/or tumorigenesis. Further studies are needed to confirm the protective effects of K71 in animal models for infectious diseases and cancer, and to elucidate the mechanisms by which K71 cell components exert immunostimulatory effects.
Conflict of interest There are no conflicts of interest to declare.
