Lactobacillus paracasei KW3110 Prevents Blue Light-Induced Inflammation and Degeneration in the Retina

Age-related macular degeneration and retinitis pigmentosa are leading causes of blindness and share a pathological feature, which is photoreceptor degeneration. To date, the lack of a potential treatment to prevent such diseases has raised great concern. Photoreceptor degeneration can be accelerated by excessive light exposure via an inflammatory response; therefore, anti-inflammatory agents would be candidates to prevent the progress of photoreceptor degeneration. We previously reported that a lactic acid bacterium, Lactobacillus paracasei KW3110 (L. paracasei KW3110), activated macrophages suppressing inflammation in mice and humans. Recently, we also showed that intake of L. paracasei KW3110 could mitigate visual display terminal (VDT) load-induced ocular disorders in humans. However, the biological mechanism of L. paracasei KW3110 to retain visual function remains unclear. In this study, we found that L. paracasei KW3110 activated M2 macrophages inducing anti-inflammatory cytokine interleukin-10 (IL-10) production in vitro using bone marrow-derived M2 macrophages. We also show that IL-10 gene expression was significantly increased in the intestinal immune tissues 6 h after oral administration of L. paracasei KW3110 in vivo. Furthermore, we demonstrated that intake of L. paracasei KW3110 suppressed inflammation and photoreceptor degeneration in a murine model of light-induced retinopathy. These results suggest that L. paracasei KW3110 may have a preventive effect against degrative retinal diseases.


Introduction
In recent years, blue light has been used in several visual display terminals (VDTs), including computers, smart phones, and tablet devices; thus, opportunities of human exposure to blue light have increased. Excessive exposure to blue light can cause photoreceptor degeneration in the retina [1] and may be related to age-related macular degeneration (AMD) [2,3] and retinitis pigmentosa [4]. AMD and retinitis pigmentosa are the leading causes of blindness in the elderly population [5]. Recently, natural compounds in foods have attracted worldwide attention in an attempt to treat light-induced ocular problems, in particular, antioxidants in foods [6][7][8]. However, the mechanism of light-induced retinal damage has not been completely elucidated.

RNA Preparation and Quantitative RT-PCR from Tissues
Total RNA was extracted from MLNs using the RNeasy Mini kit (Qiagen), and cDNAs were prepared using an iScript cDNA synthesis kit (BioRad, Hercules, CA, USA) according to the manufacturer's instructions. The resulting products were subjected to quantitative RT-PCR using SYBR Premix Ex Taq (Takara Bio, Otsu, Japan) and a LightCycler PCR system (Roche Diagnostics, Basel, Switzerland). The relative expression levels of the gene were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh). The primers used for PCR were as follows: Gapdh forward (F) (AACGACCCCTTCATTGAC) and Gapdh reverse (R) (TCCACGACATACTCAGCAC), Il10 F (CAGAGCCACATGCTCCTAGA) and Il10 R (TGTCCAGCTGGTCCTTTGTT).

Light Exposure
After acclimatization, the mice (BALB/c, male) were divided by equal average weights into three groups (n = 6). The non-light exposure control mice group and the light exposure mice group were maintained on AIN93G purified rodent diet (Zeigler, Gardners, PA, USA). In addition, the light exposure L. paracasei KW3110 mice group was fed the AIN93G diet containing approximately 1 mg heat-killed L. paracasei KW3110/day/mouse. All mice were housed in specific pathogen-free conditions under a 12-h light-dark photo cycle and had ad libitum access to water and the diet. Two weeks later, light exposure experiments were performed. Mice were exposed to blue light as previously described with slight modifications [18]. Briefly, the mice were dark-adapted for 12 h before light exposure. The mice were then exposed to 5000 lux of blue light (CCS Inc., Kyoto, Japan, peak at 470 nm) for 3 h, starting at 9:00 a.m., in exposure boxes maintained at 23 • C. After light exposure, the mice were maintained under a dim cyclic light (5 lux, 12 h on/off).

Retinal Cell Preparations
Three days after the start of light exposure, the retinas were digested with 1 mg/mL collagenase II (Worthington, Lakewood, NJ, USA) for 40 min at 37 • C in Hank's Balanced Salt Solution (HBSS) buffer with 1.0% bovine serum albumin (BSA). The tissue digest was then filtered through a 70 µm cell strainer and washed with HBSS buffer with 1.0% BSA for 5 min at 1300 rpm and at 4 • C. The supernatant was carefully removed and the digested tissue pellet was resuspended to form a single-cell suspension.

Analysis of Cytokine Concentrations
The retinal cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FCS to evaluate the production of inflammatory cytokines. Supernatants were collected and analyzed for cytokine concentrations using a Bio-Plex Pro mouse cytokine assay kit (Bio-Rad).

Measurements of the Retinal Thickness
One week after the start of light exposure, eye balls were fixed in neutral 10% formalin and decalcified. The tissues were sectioned including the regions from the optic nerve head to the most peripheral, then stained with hematoxylin and eosin. The outer nuclear layer (ONL) thickness in the retinal section was measured in all areas. We randomly selected ten observation points in each image and averaged using WinROOF software (MITANI Corporation).

Electroretinography (ERG)
After acclimatization, the mice (BALB/c, male) were divided by equal average weights into two groups. The control mice group (n = 4) was fed AIN93G diets. The L. paracasei KW3110 mice group (n = 4) was fed AIN93G containing approximately 1 mg heat-killed L. paracasei KW3110/day/mouse. All mice were housed in specific pathogen-free conditions under a 12-h light-dark (about 700 lux) photo cycle and had ad libitum access to water and the diet. Two weeks later, the mice were dark-adapted for 12 h and then placed under dim red illumination before conducting ERGs. The mice were anesthetized with an MMB combination anesthetic containing midazolam (4 mg/kg, SANDOZ, Yamagata, Japan), medetomidine (0.75 mg/kg, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) and butorphanol tartrate (5 mg/kg, Meiji Seika Pharma, Tokyo, Japan) and placed on a heating pad to maintain their body temperatures at 35-36 • C throughout the experiments. The pupils were dilated with a single drop of a mixed solution of 0.5% tropicamide and 0.5% phenylephrine (Santen Pharmaceutical, Osaka, Japan). The ground and reference electrodes were then placed on the tail and subcutaneously between the eyes, respectively, while the active gold wire electrodes were placed on the cornea. The recordings were performed with a Ganzfeld dome, an acquisition system, and LED stimulators (PuREC, MAYO Corporation, Inazawa, Japan). The amplitude of the a-wave was measured from the baseline to the trough of the a-wave. The amplitude of the b-wave was determined from trough of the a-wave to the peak of the b-wave.

Statistical Analysis
All values are presented as the mean ± SEM. Statistical differences for the results of Figure 1 were performed using Dunnett's test for post-hoc comparisons. Statistical differences between three groups (control mice group fed a control diet without light exposure, light control mice group fed a control diet with light exposure, and L. paracasei KW3110 mice group fed a diet containing L. paracasei KW3110 with light exposure) were analyzed by one-way analysis of variance (ANOVA), followed by the Tukey-Kramer test with significance set at p < 0.05. Statistical differences between the two groups (light control mice group fed a control diet with light exposure and L. paracasei KW3110 mice group fed a diet containing L. paracasei KW3110 with light exposure) were determined using an unpaired, two-tailed Student's t-test with significance set at p < 0.05. All statistical analyses were performed using the Ekuseru-Toukei 2012 software program (Social Survey Research Information, Tokyo, Japan).

L. paracasei KW3110 Activates M2 Macrophages In Vitro and Induces IL-10 Production In Vivo
In order to determine the effects of L. paracasei KW3110 on M2 macrophage activation, bone marrow-derived M-CSF-induced M2 macrophages were treated with L. paracasei KW3110 and IL-10 levels, as a marker of M2-polarization [27], were measured in culture supernatants. L. paracasei KW3110 at 0.1-10 µg/mL induced IL-10 production in a concentration-dependent manner ( Figure 1A). In the previous report, our team showed that orally provided L. paracasei KW3110 (50 mg/head) interacted with the immune cells in the gut [19]. To examine IL-10 induction of L. paracasei KW3110 in vivo, we evaluated IL-10 gene expression in mesenteric lymph nodes (MLNs) at several time points after oral administration of 50 mg/head L. paracasei KW3110 in mice. The IL-10 mRNA level in MLNs significantly increased 6 h after oral administration and decreased to the basal level 24 h after administration ( Figure 1B). These results suggest that L. paracasei KW3110 activated M2 macrophages inducing the production of IL-10.

L. paracasei KW3110 Activates M2 Macrophages in vitro and Induces IL-10 Production in vivo
In order to determine the effects of L. paracasei KW3110 on M2 macrophage activation, bone marrow-derived M-CSF-induced M2 macrophages were treated with L. paracasei KW3110 and IL-10 levels, as a marker of M2-polarization [27], were measured in culture supernatants. L. paracasei KW3110 at 0.1-10 µg/mL induced IL-10 production in a concentration-dependent manner ( Figure  1A). In the previous report, our team showed that orally provided L. paracasei KW3110 (50 mg/head) interacted with the immune cells in the gut [19]. To examine IL-10 induction of L. paracasei KW3110 in vivo, we evaluated IL-10 gene expression in mesenteric lymph nodes (MLNs) at several time points after oral administration of 50 mg/head L. paracasei KW3110 in mice. The IL-10 mRNA level in MLNs significantly increased 6 h after oral administration and decreased to the basal level 24 h after administration ( Figure 1B). These results suggest that L. paracasei KW3110 activated M2 macrophages inducing the production of IL-10.

L. paracasei KW3110 Induces Retinal M2 Macrophages Following Light Exposure
We next investigated the effects of L. paracasei KW3110 on retinal macrophages in a murine light-induced retinopathy model. Flow cytometry analyses revealed that intake of L. paracasei KW3110 significantly increased the ratio of f4/80-, CD11b-, and CD206-positive macrophages in the retina to CD11b-positive cells, 3 days after the light exposure compared with the control mice group (Figure 2A, B). We also evaluated the levels of inflammatory cytokines in retinal macrophages. Intake of L. paracasei KW3110 significantly decreased the expression of the inflammatory cytokine TNF-α in retinal macrophages compared with that in the mice group fed a control diet ( Figure 2C). In addition, the production of IL-1β ( Figure 2D left graph) and RANTES (regulated on activation, normal T cell expressed and secreted) ( Figure 2D right graph) inflammatory cytokines, were significantly lower in the mice group fed a diet containing L. paracasei KW3110 than that in the control group. These data indicate that intake of L. paracasei KW3110 induced M2 macrophages and suppressed the production of inflammatory cytokines evoked by blue light exposure.

L. paracasei KW3110 Induces Retinal M2 Macrophages Following Light Exposure
We next investigated the effects of L. paracasei KW3110 on retinal macrophages in a murine light-induced retinopathy model. Flow cytometry analyses revealed that intake of L. paracasei KW3110 significantly increased the ratio of f4/80-, CD11b-, and CD206-positive macrophages in the retina to CD11b-positive cells, 3 days after the light exposure compared with the control mice group (Figure 2A,B). We also evaluated the levels of inflammatory cytokines in retinal macrophages. Intake of L. paracasei KW3110 significantly decreased the expression of the inflammatory cytokine TNF-α in retinal macrophages compared with that in the mice group fed a control diet ( Figure 2C). In addition, the production of IL-1β ( Figure 2D left graph) and RANTES (regulated on activation, normal T cell expressed and secreted) ( Figure 2D right graph) inflammatory cytokines, were significantly lower in the mice group fed a diet containing L. paracasei KW3110 than that in the control group. These data indicate that intake of L. paracasei KW3110 induced M2 macrophages and suppressed the production of inflammatory cytokines evoked by blue light exposure.

Intake of L. paracasei KW3110 Suppresses the Photoreceptor Degeneration Induced By Light Exposure
Retinal inflammation was previously suggested to be associated with photoreceptor degeneration [10]. To evaluate the effects of L. paracasei KW3110 on light-induced retinal degeneration, we compared the ONL thickness containing photoreceptor cells from the optic nerve head to the periphery in the retina. The ONL thickness in the light-exposure mice group fed a control diet was significantly thinner than that in the non-light exposed mice fed a control diet ( Figure 3A,B). In contrast, the ONL thickness in the light-exposure mice fed a diet containing L. paracasei KW3110 was maintained at the same thickness as in the non-light exposed mice fed a control diet ( Figure 3A,B). The ONL thickness in the light-exposure mice group fed a control diet was significantly thinner than that in the light-exposed mice fed a diet containing L. paracasei KW3110 ( Figure 3A,B and Figure S1). These results indicate that intake of L. paracasei KW3110 attenuated photoreceptor degeneration caused by an excessive blue light exposure.

Intake of L. paracasei KW3110 Suppresses the Photoreceptor Degeneration Induced by Light Exposure
Retinal inflammation was previously suggested to be associated with photoreceptor degeneration [10]. To evaluate the effects of L. paracasei KW3110 on light-induced retinal degeneration, we compared the ONL thickness containing photoreceptor cells from the optic nerve head to the periphery in the retina. The ONL thickness in the light-exposure mice group fed a control diet was significantly thinner than that in the non-light exposed mice fed a control diet ( Figure 3A,B). In contrast, the ONL thickness in the light-exposure mice fed a diet containing L. paracasei KW3110 was maintained at the same thickness as in the non-light exposed mice fed a control diet ( Figure 3A,B). The ONL thickness in the light-exposure mice group fed a control diet was significantly thinner than that in the light-exposed mice fed a diet containing L. paracasei KW3110 ( Figure 3A,B and Figure S1). These results indicate that intake of L. paracasei KW3110 attenuated photoreceptor degeneration caused by an excessive blue light exposure. Values are presented as the mean ± SEM. Significance was assumed if the p value was < 0.05. *p < 0.05; **p < 0.01; non-light CTL, no light exposed mice group fed a control diet; light control, light exposed mice group fed a control diet; light KW3110, light exposed mice group fed a diet containing L. paracasei KW3110.

Intake of L. paracasei KW3110 Attenuates The Impairment of Retinal Function
To investigate the effects of intake of L. paracasei KW3110 on retinal functions, ERG analyses were performed. In the scotopic ERG, the amplitudes of the a-and b-waves tended to be lower in the mice group fed a control diet than in the mice group fed a diet containing L. paracasei KW3110 ( Figure 4A,B). In addition, the amplitude of the b-wave in the photopic ERG was significantly lower in the mice group fed a control diet than in the mice group fed a diet containing L. paracasei KW3110 ( Figure 4C). These results suggest that administration of L. paracasei KW3110 has a protective effect in both cone and rod photoreceptor functions. Values are presented as the mean ± SEM. Significance was assumed if the p value was < 0.05. *p < 0.05; **p < 0.01; non-light CTL, no light exposed mice group fed a control diet; light control, light exposed mice group fed a control diet; light KW3110, light exposed mice group fed a diet containing L. paracasei KW3110.

Intake of L. paracasei KW3110 Attenuates the Impairment of Retinal Function
To investigate the effects of intake of L. paracasei KW3110 on retinal functions, ERG analyses were performed. In the scotopic ERG, the amplitudes of the a-and b-waves tended to be lower in the mice group fed a control diet than in the mice group fed a diet containing L. paracasei KW3110 ( Figure 4A,B). In addition, the amplitude of the b-wave in the photopic ERG was significantly lower in the mice group fed a control diet than in the mice group fed a diet containing L. paracasei KW3110 ( Figure 4C). These results suggest that administration of L. paracasei KW3110 has a protective effect in both cone and rod photoreceptor functions. Values are presented as the mean ± SEM. Significance was assumed if the p value was < 0.05. *p < 0.05; **p < 0.01; non-light CTL, no light exposed mice group fed a control diet; light control, light exposed mice group fed a control diet; light KW3110, light exposed mice group fed a diet containing L. paracasei KW3110.

Intake of L. paracasei KW3110 Attenuates The Impairment of Retinal Function
To investigate the effects of intake of L. paracasei KW3110 on retinal functions, ERG analyses were performed. In the scotopic ERG, the amplitudes of the a-and b-waves tended to be lower in the mice group fed a control diet than in the mice group fed a diet containing L. paracasei KW3110 ( Figure 4A,B). In addition, the amplitude of the b-wave in the photopic ERG was significantly lower in the mice group fed a control diet than in the mice group fed a diet containing L. paracasei KW3110 ( Figure 4C). These results suggest that administration of L. paracasei KW3110 has a protective effect in both cone and rod photoreceptor functions.

Discussion
In this study, the in vitro and in vivo experiments suggested that L. paracasei KW3110 activated M2 macrophages and induced anti-inflammatory cytokine IL-10 production. We also demonstrated that L. paracasei KW3110 had a positive effect on retinal functional restoration in vivo.
In a previous study, ligands for toll-like receptor 2 (TLR2) such as Pam3Cys have been reported to activate mouse dendritic cells and induce IL-10 production through activation of the ERK pathway [28]. In the current study, lipo-teichoic acid (LTA), one of the ligands for TLR2, also slightly activated M2 macrophages and induced IL-10 production. The effect of L. paracasei KW3110 on IL-10 production in bone marrow-derived M2 macrophages might have been mediated through the TLR2-dependent ERK pathway since lactic acid bacteria have lipo-teichoic acid. In addition, peptidoglycans in lactic acid bacteria were also reported to increase IL-10 levels via the nucleotide-oligomerization domain receptor 2 (NOD2) pathway [29]. The effects of L. paracasei KW3110 on the production of IL-10 might be through the NOD2-dependent pathway at least in part.
In this study, we also showed that L. paracasei KW3110 had the potential of activating M2 macrophages and inducing the production of IL-10 in vitro and in vivo ( Figure 1A and B). Previously, we reported that orally provided L. paracasei KW3110 interacted with the gut immune cells in mice [20]. These results suggested that L. paracasei KW3110 could activate the gut immune cells inducing IL-10 production. In a previous report, oral administration of Pantoea agglomerans-derived lipopolysaccharide reduced proinflammatory cytokine expression in the blood and reduced the brain Aβ burden and memory impairment [30]. These results suggested that the regulation of cytokine levels, induced by oral administration of food constituents, might have the potential to affect the inflammatory state of the peripheral tissues through systemic blood flow. IL-10 is known as not only one of the M2 macrophage-producing anti-inflammatory cytokines but also one of the factors that induce M2 macrophages [31]. In this study, we demonstrated that intake of L. paracasei KW3110 induced CD11b-positive and CD206-positive monocytes which are generally defined as M2 type macrophages in the blue light-exposed retina ( Figure 2). Previously, blood-borne macrophages have been reported to integrate into the retina through the optic nerve and the ciliary body in a light-induced retinopathy mouse model [32]. Taken together, L. paracasei KW3110 interacts with gut immune cells and might induce M2 macrophages, at least in part, through IL-10 induced from the gut immune cells. Then, those M2 macrophages might be recruited to the retina.
M2 macrophages have been reported to have an anti-inflammatory phenotype when the tissue is damaged [33]. In this study, inflammatory macrophages, i.e., TNF-α-producing macrophages were decreased in the retina of the mice group fed a diet containing L. paracasei KW3110 compared with that of mice fed a control diet under the same blue light exposure conditions ( Figure 2C). IL-1β and RANTES, which were known as inflammatory phenotype markers in the stressed retina [34][35][36], were also significantly lower in the mice group fed a diet containing L. paracasei KW3110 (Figure

Discussion
In this study, the in vitro and in vivo experiments suggested that L. paracasei KW3110 activated M2 macrophages and induced anti-inflammatory cytokine IL-10 production. We also demonstrated that L. paracasei KW3110 had a positive effect on retinal functional restoration in vivo.
In a previous study, ligands for toll-like receptor 2 (TLR2) such as Pam3Cys have been reported to activate mouse dendritic cells and induce IL-10 production through activation of the ERK pathway [28]. In the current study, lipo-teichoic acid (LTA), one of the ligands for TLR2, also slightly activated M2 macrophages and induced IL-10 production. The effect of L. paracasei KW3110 on IL-10 production in bone marrow-derived M2 macrophages might have been mediated through the TLR2-dependent ERK pathway since lactic acid bacteria have lipo-teichoic acid. In addition, peptidoglycans in lactic acid bacteria were also reported to increase IL-10 levels via the nucleotide-oligomerization domain receptor 2 (NOD2) pathway [29]. The effects of L. paracasei KW3110 on the production of IL-10 might be through the NOD2-dependent pathway at least in part.
In this study, we also showed that L. paracasei KW3110 had the potential of activating M2 macrophages and inducing the production of IL-10 in vitro and in vivo ( Figure 1A,B). Previously, we reported that orally provided L. paracasei KW3110 interacted with the gut immune cells in mice [20]. These results suggested that L. paracasei KW3110 could activate the gut immune cells inducing IL-10 production. In a previous report, oral administration of Pantoea agglomerans-derived lipopolysaccharide reduced proinflammatory cytokine expression in the blood and reduced the brain Aβ burden and memory impairment [30]. These results suggested that the regulation of cytokine levels, induced by oral administration of food constituents, might have the potential to affect the inflammatory state of the peripheral tissues through systemic blood flow. IL-10 is known as not only one of the M2 macrophage-producing anti-inflammatory cytokines but also one of the factors that induce M2 macrophages [31]. In this study, we demonstrated that intake of L. paracasei KW3110 induced CD11b-positive and CD206-positive monocytes which are generally defined as M2 type macrophages in the blue light-exposed retina ( Figure 2). Previously, blood-borne macrophages have been reported to integrate into the retina through the optic nerve and the ciliary body in a light-induced retinopathy mouse model [32]. Taken together, L. paracasei KW3110 interacts with gut immune cells and might induce M2 macrophages, at least in part, through IL-10 induced from the gut immune cells. Then, those M2 macrophages might be recruited to the retina.
M2 macrophages have been reported to have an anti-inflammatory phenotype when the tissue is damaged [33]. In this study, inflammatory macrophages, i.e., TNF-α-producing macrophages were decreased in the retina of the mice group fed a diet containing L. paracasei KW3110 compared with that of mice fed a control diet under the same blue light exposure conditions ( Figure 2C). IL-1β and RANTES, which were known as inflammatory phenotype markers in the stressed retina [34][35][36], were also significantly lower in the mice group fed a diet containing L. paracasei KW3110 ( Figure 2D). In addition, we recently reported that L. paracasei KW3110 activated human peripheral blood mononuclear cell-(human-PBMCs) derived M2 macrophages and mitigated VDT load-induced ocular disorders, including eye fatigue, in humans [24]. These results suggested that L. paracasei KW3110 induced anti-inflammatory M2 macrophages in the stress conditioned retina.
Intake of L. paracasei KW3110 also suppressed light-induced ONL thinning (Figure 3). The ONL is composed of photoreceptor cell bodies and the ONL thickness has been reported to decrease in response to light-induced photoreceptor loss [37]. Although further studies, including analyses of apoptotic cell death of photoreceptors, are needed, intake of L. paracasei KW3110 might attenuate photoreceptor loss. We also showed that intake of L. paracasei KW3110 could mitigate the impairments of the retinal function evaluated by ERG (Figure 4). The a-wave responses as shown by scotopic ERG indicate rod photoreceptor function and the b-wave responses as shown by scotopic ERG indicate the subsequent responses of photoreceptor function. The b-wave responses as shown by photopic ERG indicate the subsequent response evoked from the cone photoreceptor function [38]. Taken together, it is suggested that intake of L. paracasei KW3110 has a protective effect in both cone and rod photoreceptor functions.
Retinal phototoxicity models in small rodents, including a mouse model of light-induced retinopathy, have been widely used in the majority of studies. However, previous studies have demonstrated that light-induced damaged retina showed various morphological patterns in different animal models [39][40][41][42]. In rats and mice, the light-induced damages in the rod photoreceptors have been reported to be more sensitive than in cone photoreceptors [43] while in chickens and pigeons cone photoreceptors have been reported to be damaged first [44]. Therefore, further studies using the larger animals are needed to confirm the preventive effects of L. paracasei KW3110 on light-induced inflammation and degeneration in the retina.

Conclusions
In summary, L. paracasei KW3110 induced retinal M2 macrophages in a murine model of light-induced retinopathy. In addition, oral intake of L. paracasei KW3110 had a positive effect on retinal morphology and function. These findings suggested that L. paracasei KW3110 might have potential as a dietary food supplement to prevent retinal degeneration through regulating inflammation in response to blue-light damage.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/10/12/1991/ s1. Figure S1: Representative images of H&E staining for retinal sections from the optic nerve head to the most peripheral area.