Lactobacillus johnsonii JERA01 upregulates the production of Th1 cytokines and modulates dendritic cells-mediated immune response

Si-Yeon Kim; Hong-Gu Joo

doi:10.4196/kjpp.24.205

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Kim and Joo: Lactobacillus johnsonii JERA01 upregulates the production of Th1 cytokines and modulates dendritic cells-mediated immune response

Original Article

Korean J. Physiol. Pharmacol. 2025;29(3):271-281.

Published online: 14 November 2024

DOI: https://doi.org/10.4196/kjpp.24.205

Lactobacillus johnsonii JERA01 upregulates the production of Th1 cytokines and modulates dendritic cells-mediated immune response

Si-Yeon Kim¹, Hong-Gu Joo^1,^2,^*

¹College of Veterinary Medicine, Jeju National University, Jeju 63243, Korea

²Veterinary Medical Research Institute, Jeju National University, Jeju 63243, Korea

^*Correspondence Hong-Gu Joo, E-mail: jooh@jejunu.ac.kr

Contributed by:

Author contributions: S.Y.K. and H.G.J. conducted experiments and performed data analysis. H.G.J. supervised and coordinated the study. S.Y.K. and H.G.J. wrote the manuscript.

Received 21 June 2024 Revised 22 September 2024 Accepted 7 October 2024

(open-access):

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Lactic acid bacteria are known to have various effects on the immune system. The type and extent of the effect differ, depending on the type of lactic acid bacteria. This study aimed to investigate the effects of Lactobacillus johnsonii bacterin on mouse-derived immune cells. Treating splenocytes with L. johnsonii bacterin slightly increased the metabolic activity. Additionally, the expression of the activation marker CD25 and production of the Th1-type inflammatory cytokine interferon (IFN)-gamma increased. We confirmed that the increase in IFN-gamma production due to L. johnsonii stimulation was mainly due to T and B cells among splenocytes. Treating dendritic cells (DCs) with L. johnsonii bacterin at concentrations of 106 and 107 cfu/ml significantly increased tumor necrosis factor-alpha, a pro-inflammatory cytokine, and interleukin-12, a cell-mediated immunity cytokine. Additionally, the expression of surface markers increased. Allogeneic mixed lymphocyte reactions showed that L. johnsonii reduced the antigen-presenting ability of DCs. In cocultures of DCs and splenocytes, L. johnsonii decreased cellular metabolic activity and increased cell death. L. johnsonii upregulated the expression of programmed death ligand 1 on DCs. The findings of this study indicate that L. johnsonii bacterin has immunomodulatory and immunostimulatory effects. While L. johnsonii increased the expression of cytokines and surface markers of immune cells, it modulated DC-mediated immune response. Further studies are needed to determine the effects of L. johnsonii bacterin on DCs and related immune cells.

Keywords: Cytokines, Dendritic cells, Immunity, Lactobacillus johnsonii, T-lymphocytes

INTRODUCTION

Proper maintenance of normal flora is strongly associated with disease prevention [1]. According to the Food Agricultural Organization/World Health Organization, probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit to the host".

Lactobacilli are distributed throughout the human and animal intestinal tract and exhibit various effects on the immune system in various ways [2]. Lactobacilli regulate the function of immune cells. Lactobacilli increase major histocompatibility complex (MHC), costimulatory adhesion, activation molecules, and interleukin (IL)-12 production. The increased IL-12 promotes the differentiation of T cells into helper T (Th)1 cells [3]. Lactobacilli inhibit activation of IgE-dependent mast cells and basophils by downregulating IgE, IL-4, and IL-5 production [4]. One of lactobacilli induces hyporesponsiveness in T cells by modulating dendritic cells (DCs) fuction [5]. This suggests that some lactobacilli modulate the function of DCs to regulate T cell differentiation and function. In this regard, there are some studies that showed positive effects of lactobacilli in alleviating autoimmune diseases such as inflammatory bowel disease [6] and allergic diseases [7].

Splenocytes comprise T lymphocytes, B lymphocytes, and natural killer (NK) cells. Upon lymphocyte activation, CD25 (IL-2 receptor) surface markers are upregulated [8]. Naïve T cells differentiate into Th1 and Th2 cells. Th1 cells secrete interferon (IFN)-gamma, which induces a cell-mediated immune response. Th2 cells secrete IL-4 and IL-5, which induce a humoral immune response. Th2 cells can play a role in activating IgE antibody-producing B cells, mast cells, and eosinophils, which induce allergic inflammation [9]. NK cells are innate immunity-associated lymphocytes that produce IFN-gamma and perform cytotoxic functions by recognizing transformed cells upon activation [10]. NK cells produce IFN-gamma in the innate immune response, and CD8⁺ cytotoxic T cells and CD4⁺ Th1 produce IFN-gamma in the adaptive immune response [11].

DCs bridge innate and adaptive immunity and are the only antigen-presenting cells that induce the activation of naive T cells. They ingest antigens and migrate to secondary lymphoid organs to present MHC-peptide complexes to T lymphocytes [12]. In this process, T cells are differentiated and activated by the proper expression of costimulatory molecules and secreted cytokines [13]. DCs are divided into myeloid and plasmacytoid DCs, and the surface marker for bone marrow-derived DCs is CD11c⁺CD123^low [14]. Upon activation, DCs produce proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and IL-12 [13]. DCs activate adaptive immunity, are involved in immune tolerance [15], and play an important role in maintaining homeostasis by being distributed throughout the gut. In the intestinal tract, which is constantly exposed to foreign antigens, intestinal DCs play a key role in regulating immune stimulation or tolerance depending on the antigen [16].

Lactobacillus johnsonii is a typical gut lactic acid bacteria. Previous studies have shown that L. johnsonii plays a positive role in activating the host's immune cells and alleviating disease symptoms [17,18]. L. johnsonii plays an important role in maintaining gut health [18]. Furthermore, L. johnsonii has been reported to enhance intestinal immunity [19], metabolic-related diseases [20], and liver disease [21]. Although there have been several studies on the effects of L. johnsonii, research on its detailed mechanism of action in immune cells is very lacking. L. johnsonii stimulates DCs to increase the expression of cytokines and modulates the differentiation of T cells to regulate the balance between T cell types [17,22]. In this study, we focused the effects of L. johnsonii on splenocytes and DC-mediated immune responses.

METHODS

Animals and reagents

C57BL/6 and Balb/c mice were purchased from SAMTAKO and housed in the animal facility at Jeju National University. In this study, 7–14-week-old mice were used, and all animal experiments were performed in accordance with the Institutional Guidelines for Animal Use and Care of Jeju National University (No.: 2023-0053). Lipopolysaccharide (LPS) was purified from Escherichia coli O26:B6 purchased from Sigma-Aldrich and diluted in sterile phosphate-buffered saline.

Preparation of L. johnsonii

L. johnsonii JERA01 used in this experiment was provided by SAMDA Co. at a concentration of 10⁹ cfu/ml. The frozen bacteria was defrosted at 36.5°C and inactivated at 90°C for 2 h in a heating block. The bacterin of L. johnsonii was diluted to a concentration of 10³–10⁷ cfu/ml.

Primary immune cells

Spleens were harvested from euthanized mice and red blood cells were hemolyzed with ammonium chloride potassium (ACK) lysis buffer (Thermo Fisher Scientific). Splenocytes were filtered through a 70 μm cell strainer and cultured in RPMI1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin-streptomycin, 1 mM sodium pyruvate, non-essential amino acids, 10 mM Hepes buffer, and 55 μM 2-mercaptoethanol. To get DCs, bone marrow cells were harvested from the femur and tibia of mice. Erythrocytes were lysed with ACK lysis buffer, passed through a 70 μm cell strainer, and cultured in RPMI 1640 medium containing 10 ng/ml of mouse recombinant granulocyte macrophage-colony stimulating factor (Peprotech), murine IL-4 (Peprotech), 5% fetal bovine serum, 100 U/ml penicillin-streptomycin, and 2 mM L-glutamine. Suspended cells were removed on days 2 and 4 of culture and semi-suspended DCs were used for experiments after 6 days. All cells were cultured at 37°C and 5% CO₂.

Measurement of cellular metabolic activity

Splenocytes were treated with L. johnsonii at different concentrations and cultured in 96-well culture plates for 3 days. To measure cell metabolic activity, cells were treated with 10 μl/well of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) and incubated at 37°C, 5% CO₂ for 4 h. In MTT assay, the wells were treated with additional 100 μl/well of 10% sodium dodecyl sulfate solution for 2 h to dissolve the formazan product (crystal violet) generated by mitochondrial reductase. Absorbance was measured at 570 nm for MTT assay using a microplate reader (Multiskan FC; Thermo Fisher Scientific).

Measurement of cytokine production

Splenocytes or DCs were treated with L. johnsonii at different concentrations and cultured in 96-well culture plates for 3 days. The supernatants were harvested and the production of IFN-gamma, TNF-alpha, or IL-12 was measured using an enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientific). Absorbance was measured at 450 nm using a microplate reader.

Flow cytometry analysis

Flow cytometry was performed to measure the expression of immune-related surface markers and activation markers of splenocytes or DCs. Splenocytes were cultured at a concentration of 2 × 10⁶ cells/ml in 24-well culture plates and treated with L. johnsonii for 3 days. Cells were washed with FACS staining solution (FSS; 5% FBS and 0.1% sodium azide in Hank's Balanced Salt Solution), followed by antibody treatment. The antibodies used were allophycocyanin (APC) anti-mouse CD3 (clone: 17A2, BioLegend), fluorescein isothiocyanate (FITC) anti-mouse/human CD45R/B220 (clone: RA3-6B2, BioLegend), FITC anti-mouse NK 1.1 (clone: S17016D, BioLegend), and phycoerythrin (PE) anti-mouse CD25 (clone: 3C7, BioLegend). DCs were cultured at a concentration of 1 × 10⁵ cells/ml in 24-well culture plates and treated with L. johnsonii or LPS for 3 days. Cells were washed with FSS and then treated with FITC anti-mouse MHC class II (I-A^b, clone: AF6-120.1, BioLegend), Alexa Fluor 700 anti-mouse CD86 (clone: GL-1, BioLegend), programmed death ligand 1 (PD-L1, clone: 10F.9G2, BioLegend), FasL (clone: MFL3, BioLegend) and APC anti-mouse CD11c (clone: N418, BioLegend) antibodies. TruStain FcX anti-mouse CD16/32 (clone: 93, BioLegend) was used to prevent non-specific reactions. After antibody staining, all cells were washed twice with FSS and flow cytometrically analyzed using a CytoFLEX LX flow cytometer (Beckman Coulter) and CytExpert software (Beckman Coulter).

Mixed lymphocyte reaction (MLR)

For the allogeneic MLR assay, C57BL/6 mouse-derived DCs and Balb/c mouse-derived splenocytes were co-cultured. The cells were cultured in 96-well culture plates at a concentration of 1 × 10⁵ cells/ml and 2 × 10⁶ cells/ml, respectively, and were treated with L. johnsonii at different concentrations and incubated for 5 days. To determine the activity of splenocytes stimulated by DCs, 10 μl/well of the Cell Counting Kit-8 (CCK-8, DOJINDO Laboratories) solution was treated and the absorbance was measured at 450 nm after incubation at 37°C for 4 h. Furthermore, IFN-gamma production was measured using ELISA.

Measurements of cell death

The cells of MLR were stained with annexin V-FITC and propidium iodide (PI) solution to measure cell death. DCs and allogeneic splenocytes were co-cultured with L. johnsonii in 96-well culture plates for 5 days. At the end of the incubation, the cocultured cells were washed with annexin V binding buffer (Thermo Fisher Scientific) and treated with 1 μl annexin V-FITC (BioLegend) for 10 min at room temperature and dark. PI solution was added at a concentration of 0.25 μg/ml at 5 min before the assay. Flow cytometry was performed using a CytoFLEX LX flow cytometer (Beckman Coulter). For trypan blue staining, the cells were stained with 0.4% trypan blue solution (Thermo Fisher Scientific). The number of live and dead cells was counted using a hemocytometer.

Intracellular cytokine staining

Splenocytes were cultured at a concentration of 2 × 10⁶ cells/ml in 96-well culture plates and treated with L. johnsonii (10⁵–10⁷ cfu/ml) for 5 days. Each well was treated with 10 ng/ml phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 1 ng/ml ionomycin calcium salt (Sigma-Aldrich) and incubated for 3 h. To prevent the secretion of the cytokines intracellularly produced, 10 µg/ml brefeldin A (Sigma-Aldrich) was added to at 2 h before harvesting. Harvested cells were washed twice with FSS and stained for intracellular cytokines using the Cytofix/Cytoperm kit (BD pharmingen) according to the manufacturer's instructions. Antibodies used were APC anti-mouse CD3 (clone: 17A2, BioLegend), FITC anti-mouse/human CD45R/B220 (clone: RA3-6B2, BioLegend), FITC anti-mouse NK 1.1 (clone: S17016D, BioLegend), and PE anti-mouse IFN-gamma (BD pharmingen). TruStain FcX anti-mouse CD16/32 (clone: 93, BioLegend) was used to prevent non-specific reactions. Flow cytometry was performed using a CytoFLEX LX flow cytometer (Beckman Coulter) and CytExpert software (Beckman Coulter).

Statistical analysis

Statistical analysis was performed using Prism 9 (GraphPad Software), and experimental results were expressed as mean ± standard deviation. Significance was determined using Dunnett's multiple comparison test of ordinary one-way ANOVA or ordinary two-way ANOVA. *, **, ***, and **** indicate significance levels of p < 0.05, 0.01, 0.001, and 0.0001 compared to the control, respectively.

RESULTS

L. johnsonii increases the metabolic activity of splenocytes

The MTT assay was performed to determine the effect of L. johnsonii bacterin on the metabolic activity of splenocytes (Fig. 1). The metabolic activity of splenocytes treated with L. johnsonii at a concentration range of 10⁴ –10⁷ cfu/ml significantly increased compared with control (L. johnsonii 0 cfu/ml). L. johnsonii at the same concentrations was incubated in the medium only, and absorbance was measured to confirm the negative control without splenocytes.

L. johnsonii increases surface marker expression and cytokine production in splenocytes

The expression of surface markers on splenocytes was measured using flow cytometry to determine whether L. johnsonii bacterin induces splenocyte activation (Fig. 2A). L. johnsonii at a concentration range of 10⁵–10⁷ cfu/ml consistently increased the ratio of T lymphocytes compared with the control. The expression of the CD25 surface marker, which indicates the activation of T lymphocytes, B lymphocytes, and NK cells, increased with an increase in the concentrations of L. johnsonii. IFN-gamma production was measured using ELISA to determine whether L. johnsonii bacterin affects the cytokine production of splenocytes (Fig. 2B). At concentrations up to 10⁵ cfu/ml of L. johnsonii, IFN-gamma production was similar to that of the control (0 cfu/ml). However, at concentrations above 10⁶ cfu/ml, IFN-gamma production was significantly increased compared with the control and markedly increased at a concentration of 10⁷ cfu/ml.

L. johnsonii increases IFN-gamma production by T lymphocytes and B lymphocytes

Intracellular cytokine staining was performed after culturing L. johnsonii-treated splenocytes for 5 days to determine which subsets of L. johnsonii-stimulated splenocytes contribute to increased IFN-gamma production (Fig. 3). Flow cytometry showed increased IFN-gamma production by T lymphocytes, B lymphocytes, and NK cells at concentrations above 10⁶ cfu/ml of L. johnsonii. However, the increase in IFN-gamma production by NK cells was very low. L. johnsonii mainly stimulated T and B lymphocytes. The positive control, LPS increased IFN-gamma production by B lymphocytes compared with other lymphocyte subsets.

L. johnsonii increases surface marker expression and cytokine production in DCs

Changes in surface marker expression on DCs were examined by flow cytometry to determine whether L. johnsonii bacterin induced maturation and activation of DCs (Fig. 4A). The proportion of CD86^high/MHCII^high DCs, indicative of activated DCs, increased by 23% at an L. johnsonii concentration of 10⁶ cfu/ml and by 38% at an L. johnsonii concentration of 10⁷ cfu/ml compared with the control. The highest percentage of CD86^high/MHCII^high DCs was measured when treated with the positive control LPS (0.1 μg/ml). The production of TNF-alpha and IL-12 was measured by ELISA to determine whether L. johnsonii bacterin affects the cytokine production of DCs (Fig. 4B). TNF-alpha production was not significantly different from the control at concentrations of up to 10⁵ cfu/ml L. johnsonii and increased at concentrations above 10⁶ cfu/ml. Particularly, a significant increase in TNF-alpha production was observed at a concentration of 10⁷ cfu/ml. IL-12 production was similar to that of TNF-alpha. The positive control LPS (0.1 μg/ml) increased the TNF-alpha and IL-12 production by DCs.

L. johnsonii reduced the antigen-presenting ability of DCs

Allogeneic MLR was performed to determine the effect of L. johnsonii bacterin on the antigen-presenting ability of DCs. DCs from C57BL/6 mice and splenocytes from Balb/c mice were cocultured to perform allogeneic MLR and treated with different concentrations of L. johnsonii for 5 days. The metabolic activity of cocultured cells was measured using the CCK-8 assay (Fig. 5A). The metabolic activity of cocultured cells significantly decreased at L. johnsonii concentrations above 10⁵ cfu/ml compared with the control. To analyze cell death, flow cytometry was performed after annexin V-FITC/PI staining (Fig. 5B). Necrosis (annexin V-/PI+), early apoptosis (annexin V+/PI-), and late apoptosis (annexin V+/PI+) were categorized. Compared to the control, there was a 16.1% increase in early apoptosis and a 10.1% increase in late apoptosis at 10⁷ cfu/ml. Trypan blue staining was also performed to confirm cell death. At a concentration of L. johnsonii 10⁷ cfu/ml, cell viability was significantly reduced compared to the control (data not shown).

L. johnsonii increases the expression of PD-L1 on DCs

To search the molecules that have a negative effect on the interaction between splenocytes and DCs, we checked the expression of PD-L1 and FasL by flow cytometry. The DCs were treated with L. johnsonii for 3 days. The expression of PD-L1 was marginally increased by L. johnsonii treatment (Fig. 6), indicating that L. johnsonii may influence the antigen-presenting ability of DCs by modulating the expression of PD-L1. On the other side, the expression of FasL on L. johnsonii-treated DCs was not altered (data not shown).

DISCUSSION

Lactic acid bacteria modulate the immune system of the host, which influences the composition of the gut microbiome [21]. Previous studies have shown that the expression of MHC II on DCs stimulated by L. johnsonii is increased and that monocyte-derived DCs exposed to L. johnsonii modulate the differentiation of T cells [17]. This study showed that L. johnsonii has both immunostimulatory and immunomodulatory effects. L. johnsonii increased the expression of Th1 cytokines in immune cells and induced the cell death of cocultured splenocytes with DCs.

Regarding the immunostimulatory effects, L. johnsonii increased the metabolic activity of splenocytes in a concentration-dependent manner and the expression of surface markers, indicating that the overall lymphocyte number and activation increased (Figs. 1 and 2A). Furthermore, an increase in the production of IFN-gamma was observed at L. johnsonii concentrations above 10⁶ cfu/ml (Fig. 2B). L. johnsonii increased the expression of MHC class II and CD86 molecules on DCs and Th1 cytokines, TNF-alpha and IL-12 (Fig. 4B).

L. johnsonii significantly increased the production of Th1 cytokines (IFN-gamma, TNF-alpha, and IL-12) in immune cells (Figs. 2B and 4B). Intracellular cytokine staining was performed to determine which lymphocytes contribute to increased IFN-gamma production among splenocytes. This method accumulates cytokines within cells to increase detection. Brefeldin A inhibits the movement of proteins in cells from the endoplasmic reticulum to the Golgi complex, causing protein accumulation in the endoplasmic reticulum [23]. Accumulated cytokine production can be measured by increasing the permeability of the cell membrane. The results showed that L. johnsonii-stimulated splenocytes, mainly T and B lymphocytes, contributed to increased IFN-gamma production (Fig. 3). Previous studies have shown that lactic acid bacteria are involved in the balance between Th1 and Th2 cells. This effect has been confirmed in atopic dermatitis [24], and an asthma model. Furthermore, L. johnsonii has been shown to increase the production of Th1 cytokines and decrease the production of Th2 cytokines, thus maintaining the balance between Th1 and Th2 cells [25]. These findings indicate that certain components of L. johnsonii bacterin enhance IFN-gamma production in T and B lymphocytes and may induce the conversion of Th0 cells into Th1 cells.

Regarding the immunomodulatory effects, the antigen-presenting ability of DCs was confirmed using allogeneic MLR. L. johnsonii decreased the metabolic activity and increased the cell death of the coculture, indicating that L. johnsonii reduced the antigen-presenting ability of DCs (Fig. 5). We confirmed the increased expression of PD-L1 on L. johnsonii-treated DCs (Fig. 6). Binding of B7 on antigen presenting cells to CD28 on naive T cells provides a co-stimulatory signal to T cells, resulting in IL-2 production and CD25 expression. B7 binding to cytotoxic T-lymphocyte associated protein 4 results in immunosuppression, which inhibits IL-2 production and terminates the T cell response [26]. PD-1 is one of the inhibitory receptors expressed on T cells and is expressed upon T cell activation. When PD-1 binds to PD-L1 on antigen presenting cells, it causes CD28 pathway inhibition, which inhibits T cell function and induces T cell death. Activation of DCs or elevated IFN-gamma increases the expression of PD-L1 [26,27]. Therefore, it can be considered that the activation of DCs by L. johnsonii increased the expression of PD-L1 and induced lymphocyte death. One of the death programs of T cells is activation-induced cell death (AICD). This is often caused by a death ligand, FasL [28]. We considered AICD as one of the mechanisms by which cocultured cells exposed to L. johnsonii were killed. However, there was no significant change in the expression of FasL. Further studies are needed to determine which types of lymphocytes are killed by L. johnsonii exposure in coculture and whether there are additional mechanisms that trigger cell death.

Gut microbiota can affect immune organs located away from the gut. Oral administration of lactic acid bacteria can affect the immune system of the skin [24]. Additionally, it is involved in the regulation of immune cells in the lungs via the gut-lung axis [29,30]. Considering that L. johnsonii has immunostimulatory and immunomodulatory effects, further studies are needed to determine how L. johnsonii affects the gut and other organs in relation to the immune system in vivo.

In conclusion, this study indicates that L. johnsonii bacterin affects the regulation of immune homeostasis in the host. L. johnsonii increased Th1-type cytokines and identified T and B lymphocytes as the main IFN-gamma-producing cells. On the other side, L. johnsonii showed immunomodulatory effects by reducing antigen-presenting ability. Further studies are needed to understand why L. johnsonii has two opposing effects on immunity. L. johnsonii bacterin may contain a mixture of substances that induce immunostimulatory and immunomodulatory effects. It is possible that cellular components of L. johnsonii exert immunostimulatory and immunomodulatory effects through toll-like receptors (TLRs). TLRs are involved in the maturation of DCs, which affects the differentiation and function of T cells [31]. Previous research has also shown that L. johnsonii upregulated Th1 cytokines and inflammatory cytokines in DCs while also stimulating the upregulation of regulatory T cell-type anti-inflammatory cytokines [17]. It is suggested that L. johnsonii mediates immune balance responses among immune cells. In this study, we demonstrated the immunostimulatory and immunomodulatory effects of L. johnsonii, providing a basis for further research and clinical applications of L. johnsonii for its health benefits.

ACKNOWLEDGEMENTS

None.

Notes

FUNDING

This research was financially supported by the Ministry of Trade, Industry and Energy, Korea, under the “Regional Innovation Cluster Development Program (R&D, P0025271)” supervised by the Korea Institute for Advancement of Technology (KIAT).

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

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Fig. 1

Metabolic activity of splenocytes treated with

Lactobacillus johnsonii. Splenocytes were cultured in 96-well culture plates and incubated with various concentrations of L. johnsonii (0–10⁷ cfu/ml). After 3 days, MTT assay was performed to measure cellular metabolic activity. Absorbance was measured at 570 nm using a microplate reader. As a negative control, only L. johnsonii was treated at each concentration without cells in the medium. Statistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparisons test. ***p < 0.001 and ****p < 0.0001. Lj, Lactobacillus johnsonii.

Fig. 2

Lactobacillus johnsonii activates splenocytes and increases cytokine production of splenocytes. (A) Splenocytes were cultured in 24-well culture plates and treated with L. johnsonii. After 3 days, splenocytes are stained for surface markers. Stained cells were analyzed using flow cytometry. (B) Splenocytes were treated with L. johnsonii for 3 days and the production of IFN-gamma was measured using ELISA. Absorbance was measured at 450 nm using a microplate reader. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. **p < 0.01 and ****p < 0.0001. Lj, Lactobacillus johnsonii; IFN, interferon.

Fig. 3

Lactobacillus johnsonii induces intracellular IFN-gamma production of splenocytes. Splenocytes (2 × 10⁶ cells/ml) were treated with L. johnsonii and cultured for 5 days, then cells were stimulated for last 5 h. LPS (0.1 μg/ml) was treated as a positive control. The intracellular cytokine staining was performed for IFN-gamma. Stained cells were analyzed using flow cytometry. The plotted cells are gated as CD3, B220, NK1.1 cells. Lj, Lactobacillus johnsonii; IFN, interferon; LPS, lipopolysaccharide.

Fig. 4

Lactobacillus johnsonii activates DCs and increases cytokine production of DCs. (A) DCs were cultured in 6-well culture plates and treated with L. johnsonii or LPS (0.1 μg/ml) for 3 days. Cells are stained for surface markers and analyzed using flow cytometry. (B) DCs were cultured in 96-well culture plates and treated with L. johnsonii for 3 days and the production of TNF-alpha and IL-12 was measured using ELISA. Absorbance was measured at 450 nm using a microplate reader. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. **p < 0.01 and ****p < 0.0001. DCs, dendritic cells; Lj, Lactobacillus johnsonii; LPS, lipopolysaccharide; TNF, tumor necrosis factor; IL, interleukin; MHC, major histocompatibility complex.

Fig. 5

Lactobacillus johnsonii decreases the antigen-presenting ability of DCs. DCs and allogeneic splenocytes were co-cultured at a concentration of 1 × 10⁵ cells/ml and 2 × 10⁶ cells/ml, respectively. In the coculture, L. johnsonii was treated at the indicated concentrations. (A) At 5 day, CCK-8 assay was performed. CCK-8 solution was added, and the optical density was measured at 450 nm using a microplate reader. (B) At 5 day, annexin V-FITC/PI staining was performed. The quadrants of the dot plot indicate live cells (annexin V-/PI-), cells in early apoptosis (annexin V+/PI-), late apoptosis (annexin V+/PI+), and necrosis (annexin V-/PI+). Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05, ***p < 0.001, and ****p < 0.0001. DCs, dendritic cells; Lj, Lactobacillus johnsonii; CCK-8, Cell Counting Kit-8; PI, propidium iodide.

Fig. 6

Lactobacillus johnsonii increases the expression of PD-L1 on DCs. DCs were cultured in 6-well culture plates (1 × 10⁵ cells/ml) and treated with L. johnsonii at the indicated concentrations. The cells were stained with PD-L1 antibody and analyzed using flow cytometry. (A) A representative data set was presented. The number in the histograms indicates mean fluorescence intensity (MFI). (B) Relative MFI values are normalized to the control group, which is set to 100%. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. **p < 0.01. PD-L1, programmed death ligand 1; DCs, dendritic cells; Lj, Lactobacillus johnsonii; ns, not significant (p > 0.05).

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