Lactic acid bacterium, Lactobacillus paracasei KW3110, suppresses inflammatory stress-induced caspase-1 activation by promoting interleukin-10 production in mouse and human immune cells

A strain of lactic acid bacteria, Lactobacillus paracasei KW3110 (KW3110), activates M2 macrophages with anti-inflammatory reactions and mitigates aging-related chronic inflammation and blue-light exposure-induced retinal inflammation in mice. However, the mechanism underlying the anti-inflammatory effects of KW3110 remains unclear. In this study, we investigated the anti-inflammatory effects of KW3110 using both mouse and human immune cells and evaluated the suppressive effect of KW3110 on the inflammatory reactions of the cells stimulated with lipopolysaccharide and adenosine 5′-triphosphate (LPS/ATP). KW3110 treatment induced anti-inflammatory cytokine interleukin (IL)-10 production in the supernatants of murine macrophage-like cells, J774A.1, and suppressed IL-1β production in the supernatants of LPS/ATP-stimulated cells. The influence of KW3110 on the production of these cytokines was inhibited by pre-treatment with phagocytosis blocker or transfection with siRNAs for IL-10 signaling components. KW3110 treatment also suppressed activation of caspase-1, an active component of inflammasome complexes, in LPS/ATP-stimulated J774A.1 cells, and its effect was inhibited by transfection with siRNAs for IL-10 signaling components. In addition to the effects of KW3110 on J774A.1 cells, KW3110 treatment induced IL-10 production in the supernatants of human monocytes, and KW3110 or IL-10 treatment suppressed caspase-1 activation and IL-1β production in the supernatants of LPS/ATP-stimulated cells. These results suggest that KW3110 suppresses LPS/ATP stimulation-induced caspase-1 activation and IL-1β production by promoting IL-10 production in mouse and human immune cells. Our findings reveal a novel anti-inflammatory mechanism of LAB and the effect of KW3110 on caspase-1 activation is expected to contribute to constructing future preventive strategies for inflammation-related disorders using food ingredients.


Introduction
The immune response is a fundamental system developed in animals to combat endogenous or exogenous antigens or other stresses. Inflammation is an important process in the immune response as it eliminates infectious factors and damaged cells and tissues, initiating tissue repair. Various stresses, such as some infections or aging, can cause excessive or chronic inflammation, leading to serious disorders or diseases and this is affecting more and more people [1][2][3][4]. Interventions to prevent the conditions related to those stresses are necessary.
Food ingredients with immune modulating functions have been studied as one of the strategies to prevent inflammation-related disorders or diseases [5][6][7]. Lactic acid bacteria (LAB) are well known sources of probiotics and paraprobiotics which improve gut barrier function and the immune system. Some LAB strains are reported to ameliorate several inflammationrelated disorders, such as allergies and metabolic disorders [8,9]; however, the mechanism of action is not completely understood.
We previously reported that a specific strain of LAB, Lactobacillus paracasei KW3110 (KW3110), suppresses excessive inflammation including dermatitis in mice and humans [8,[10][11][12]. KW3110 was shown to activate M2 macrophages with anti-inflammatory reactions, and induce the production of anti-inflammatory cytokine interleukin (IL)-10 [13]. In addition, KW3110 mitigated aging-related chronic inflammation and blue-light exposure-induced retinal inflammation in mice, at least partially through activating immune cells and reducing proinflammatory cytokine production, such as IL-1β [13,14]; however, the mechanism underlying the anti-inflammatory effects of KW3110 remains unclear.
To investigate the detailed site of action of the anti-inflammatory effects of KW3110, we performed in vitro experiments with murine macrophage-like cell line, J774A.1 which is a wellestablished cell line producing pro-or anti-inflammatory cytokines in response to various stimulations [15,16]. Since human monocytes have been used to investigate inflammatory reactions and their mechanism in human macrophages, we also used a human cell line to determine whether the mechanisms of anti-inflammation are preserved between murine and human cells [17,18]. Our study demonstrated that KW3110 suppressed inflammatory stress-induced activation of an active component of inflammasome complexes, caspase-1, and production of IL-1β by promoting IL-10 production in murine macrophages and human monocytes. These findings reveal a novel anti-inflammatory mechanism of LAB and are expected to contribute to constructing future preventive strategies for inflammation-related disorders using food ingredients.

Preparation of human monocytes
Human peripheral blood mononuclear cells from healthy donors were purchased from iQ Biosciences (Berkeley, CA, USA; catalog number: IQB-PBMC103). Written consent was obtained from all donor volunteers participating in a donor program that was approved by Western Institutional Review Board, and de-identification was performed. Monocytes were isolated from the mononuclear cells using a classical monocyte isolation kit (Miltenyi Biotec, Sunnyvale, CA, USA), according to the manufacturer's instructions.

Determination of cytokine production
Cells were seeded and incubated overnight in 24-well plates (J774A.1 cells, 1 × 10 5 cells/well; human monocytes, 2.5 × 10 5 cells/well), and treated with KW3110 (1.25-5 μg/mL for J774A.1 cells and 100 μg/mL for human monocytes) for 24 h at 37˚C. To measure IL-1β, the cells were treated with 1 μg/mL of IL-10 or primed with 10 μg/mL of LPS for 4 h, and stimulated with 2 mM ATP for 1 h. For the supernatant transferal experiments, in Fig 1B, the supernatants collected from KW3110-treated J774A.1 cells were transferred to other cells which were then primed with 10 μg/mL of LPS for 4 h and stimulated with 2 mM ATP for 1 h. For the phagocytosis blocking experiments, 1 μg/mL of cytochalasin D was added 30 min prior to KW3110

Western blotting
J774A.1 cells were seeded and incubated overnight in 24-well plates (1 × 10 5 cells/well), and treated with 5 μg/mL of KW3110 in Opti-MEM with 1% fetal bovine serum (Gibco, Grand Island, NY, USA) for 24 h at 37˚C. The cells were then primed with 10 μg/mL of LPS for 4 h, and stimulated with 2 mM ATP for 1 h. After stimulation, cells were lysed with radioimmunoprecipitation assay buffer and whole cell lysates were prepared and then boiled in sodium dodecyl sulfate (SDS) sample buffer for 5 min at 95˚C. Supernatants were precipitated with methanol/chloroform and the pellets were lysed in the SDS sample buffer and then boiled for 5 min at 95˚C. SDS-polyacrylamide gel electrophoresis-separated samples were transferred to polyvinylidene difluoride membranes and immunoblotted with primary antibodies (1:1000 dilution) overnight at 4˚C, followed by secondary antibodies (1:10000 dilution) for 1 h at room temperature. The membranes were scanned with an ImageQuant LAS 4000 (GE Healthcare, Chicago, IL, USA).

Caspase-1 activity assay
Cells were seeded and incubated overnight in 24-well plates (J774A.1 cells, 1 × 10 5 cells/well; human monocytes, 2.5 × 10 5 cells/well) before treatment with KW3110 (5 μg/mL for J774A.1 cells and 100 μg/mL for human monocytes) for 24 h at 37˚C. The cells were then treated with 1 μg/mL of IL-10 or primed with 10 μg/mL of LPS for 4 h, and stimulated with 2 mM ATP for 1 h. These supernatants were collected and centrifuged at 5,000 rpm for 2 min. Caspase-1 activity levels were measured with a Caspase-Glo 1 Inflammasome assay kit (Promega, Madison, WI, USA), according to the manufacturer's instructions.

Statistical analysis
Values indicate means ± SEM. Statistical differences were analyzed by unpaired t-test or analysis of variance (ANOVA) followed by Dunnett's or Tukey's test. P values < 0.05 were considered statistically significant. Experiments of evaluating cytokine production and caspase-1 activity were performed at least twice independently.

KW3110 treatment suppresses IL-1β production in LPS/ATP-stimulated J774A.1 cells
We investigated how KW3110 suppresses the inflammatory response in macrophages using a murine macrophage-like cell line, J774A.1. IL-1β levels were significantly increased in the supernatant from LPS/ATP-stimulated J774A.1 cells when compared with the control (P < 0.001; Fig 1A); KW3110 pre-treatment of the cells dose-dependently decreased IL-1β levels (5 μg/mL, P = 0.027; Fig 1A). In addition, IL-1β levels were significantly decreased in the supernatant from LPS/ATP-stimulated cells after treatment with the supernatant from KW3110-treated cells (P = 0.003; Fig 1B).

Phagocytosis is essential for KW3110-induced IL-10 production and IL-1β suppression in J774A.1 cells
Macrophages detect and phagocytose microbes including LAB, producing pro-or anti-inflammatory cytokines [21,22]. To investigate whether KW3110 activates macrophages and promotes IL-10 production through phagocytosis, we used a phagocytosis blocker, cytochalasin D, before treatment of J774A.1 cells with KW3110. Cytochalasin D pre-treatment significantly decreased both mRNA and protein levels of IL-10 in KW3110-treated cells (P < 0.001; Fig 3A) (P < 0.001; Fig 3B). Furthermore, the suppressive effect of KW3110 on IL-1β production in the supernatant from LPS/ATP-stimulated cells was significantly inhibited by cytochalasin D pre-treatment (P < 0.001; Fig 3C).

KW3110 suppresses the active form of caspase-1 expression in LPS/ATPstimulated J774A.1 cells through IL-10 signaling
We investigated the detailed mechanism of how KW3110 suppresses IL-1β production in LPS/ ATP-stimulated J774A.1 cells. The suppression of mature IL-1β expression in the supernatant from KW3110-and LPS/ATP-stimulated cells was confirmed by western blotting, whereas pro-IL-1β expression levels in the cell lysates was not affected by KW3110 treatment (Fig 4A). Since IL-1β maturation is promoted by the activation of inflammatory cysteine protease caspase-1 in inflammasome complexes, we investigated whether KW3110 suppresses caspase-1 activation in macrophages. Levels of the active form of caspase-1 were increased in the supernatant from LPS/ATP-stimulated cells when compared with the control, whereas KW3110 pre-treatment of the cells suppressed caspase-1 levels ( Fig 4B). Moreover, the suppressive effect of KW3110 on the active form of caspase-1 was inhibited by transfection with IL-10 or IL-10 receptor α siRNAs (Fig 4B).

Effects of KW3110 treatment on caspase-1 activity and IL-1β production in human monocytes
Finally, we investigated whether KW3110 treatment suppresses caspase-1 activity and IL-1β production through the IL-10 signaling pathway in human monocytes, isolated from human peripheral blood mononuclear cells used for inflammasome assay, as well as J774A.1 cells. KW3110 treatment significantly increased IL-10 levels in the supernatant from human monocytes when compared with the control (P < 0.001; Fig 6A). While LPS/ATP stimulation significantly increased IL-1β levels in cell supernatants (P < 0.001; Fig 6B), KW3110 pre-treatment significantly suppressed the production of IL-1β (P < 0.001; Fig 6B). Furthermore, caspase-1 activity levels were significantly increased by LPS/ATP stimulation of human monocytes

Discussion
Previously, a specific strain of LAB, KW3110, was shown to activate M2 macrophages and have anti-inflammatory effects, including suppression of IL-1β production, but the underlying mechanism was not well understood. Our study demonstrated that KW3110 suppressed LPS/ ATP stimulation-induced caspase-1 activation and IL-1β production by promoting IL-10

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production in murine macrophages. In addition, we showed that the mechanism of action may be similar in human monocytes.
In this study, some of the molecules released from KW3110-stimulated J774A.1 cells were shown to suppress IL-1β production in LPS/ATP-stimulated cells (Fig 1B). IL-10 production was upregulated in the supernatant from KW3110-stimulated cells (Fig 2A), and knockdown of IL-10 signaling components using siRNAs inhibited the suppressive effect of KW3110 on IL-1β production in LPS/ATP-stimulated cells (Fig 2E). In addition, IL-10 production was upregulated in the supernatant from KW3110-stimulated human monocytes (Fig 6A), and both KW3110 and recombinant IL-10 protein treatments suppressed IL-1β production in LPS/ATP-stimulated cells (Fig 6B). IL-10 is a representative anti-inflammatory cytokine and is reported to be involved in inhibition of IL-1β production [23][24][25], which is consistent with our results shown in Fig 2B. KW3110 was previously reported to activate macrophages by interacting with gut immune cells and promoting cytokine production, including IL-10 [10,13,20]. It has also been reported that KW3110 suppresses the level of pro-inflammatory cytokines such as IL-1β in the serum of aged mice or the retina of blue-light exposure-induced mice [13,14]. These results suggest that KW3110 ameliorates IL-1β production in inflammatory-stressed immune cells through the IL-10 signaling pathway in both mice and humans (Fig 7). The cells were transfected with IL-10 or IL-10 receptor α siRNAs and treated with KW3110 (10 μg/mL) for 24 h, and then treated with LPS (10 μg/mL) for 4 h and ATP (2 mM) for 1 h. The supernatant and cell lysate were analyzed by western blotting to detect caspase-1 (p20), pro-caspase-1, and β-actin. The black arrows are pointing to bands representing matured IL-1β or caspase-1 (p20). All blot images are spliced along with black lines. Sup; supernatant, Cell; cell lysate, siNT; siRNA for non-targeting, siIL-10; siRNA for IL-10, siIL-10R; siRNA for IL-10 receptor α, mIL-1β; mature IL-1β.

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The effects of KW3110 on both induction of IL-10 production and suppression of IL-1β production in J774A.1 cells were inhibited by cytochalasin D, a phagocytosis blocker (Fig 3). These results are consistent with previous reports showing that microbes, including LAB, are detected and phagocytosed by immune cells such as macrophages, followed by production of pro-or anti-inflammatory cytokines [21,22]. We suggest that KW3110 has anti-inflammatory effects which are dependent upon the phagocytosis process of macrophages. A variety of innate immune receptors including scavenger receptors, C-type lectins and Toll-like receptors are expressed in the cell membrane of macrophages and play a key role in recognizing some of the structures on the bacterial surface, thus facilitating phagocytosis [26,27]. Therefore, the antiinflammatory effect of KW3110 on macrophages might be achieved through recognition of KW3110 surface structures by macrophage receptors and the phagocytosis process.

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KW3110 treatment suppressed mature IL-1β expression in the supernatants from LPS/ ATP-stimulated J774A.1 cells, but not pro-IL-1β expression in the cell lysates (Fig 4A), indicating that KW3110 may affect the maturation process of IL-1β. IL-1β maturation is promoted by the activity of caspase-1, which is an active component of inflammasome complexes [28]. In our study, KW3110 treatment reduced caspase-1 activation in the supernatant from LPS/ATPstimulated J774A.1 cells and this effect was inhibited by transfection with siRNAs for IL-10 or IL-10 receptor α (Figs 4B and 5A). In addition, IL-10 treatment reduced caspase-1 activation in the supernatant from LPS/ATP-stimulated cells (Fig 5B), which is supported by previous reports showing that IL-10 is involved in the inhibition of inflammasome activation [29,30]. Inflammasome activation is promoted by the production of mitochondrial reactive oxygen species and oxidized mitochondrial DNA derived from mitochondrial dysfunction [31,32]. The effect of IL-10 on inhibiting inflammasome activation was suggested to be dependent on mitophagy induction, which eliminates dysfunctional mitochondria; KW3110 might induce mitophagy through the IL-10 signaling pathway [25]. In addition, these results suggest that KW3110 may mitigate inflammasome activation in inflammatory-stressed macrophages through the IL-10 signaling pathway.
In conclusion, our results demonstrate that KW3110 suppresses LPS/ATP stimulationinduced caspase-1 activation and IL-1β production by promoting IL-10 production in mouse

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and human immune cells. The suppressive effect of KW3110 on caspase-1 activation is a novel anti-inflammatory mechanism of LAB. These findings are expected to contribute to constructing future preventive strategies for inflammation-related disorders using food ingredients.