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Supplementation with oral probiotic bacteria maintains cutaneous immune homeostasis after UV exposure

European Journal of Dermatology. Volume 16, Number 5, 511-7, September-October 2006, Investigative report

DOI : 10.1684/ejd.2006.0023

Summary

Author(s) : Audrey Guéniche, Jalil Benyacoub, Timo M Buetler, Hans Smola, Stephanie Blum , Nestlé Research Center, Vers-chez-les-Blanc, PO Box 44, CH-1000, Lausanne 26, Switzerland.

Summary : Probiotic bacteria have been shown to modulate the immune system of the gut and protect against infectious and inflammatory diseases of the gastro-intestinal tract. Ultraviolet radiation (UVR) is known to alter the cutaneous and systemic immune systems implicated in the development of skin tumors. In this study we investigated whether oral probiotics are able to modulate the immune system of the skin using hairless Skh:hr1 mice exposed to an acute dose of UVR. We show that nutritional supplementation with Lactobacillus johnsonii (La1) at 10 ⁸ cfu/day for 10 days was able to protect against the UVR-induced suppression of contact hypersensitivity, the decreased epidermal Langerhans cell density and the increased IL-10 serum levels. In the absence of UV exposure, probiotic bacteria had no detectable effect on the immune system of the skin, thus acting only to re-establish skin homeostasis. In conclusion, our data demonstrate that ingested probiotic bacteria can maintain cutaneous immune capacity after UV exposure.

Keywords : cfu, colony forming unit, CHS, contact hypersensitivity, DNFB, 2,4-dinitrofluorobenzene, LC, Langerhans cell, MED, minimal erythema dose, UVR, ultraviolet radiation

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ARTICLE

Auteur(s) : Audrey Guéniche, Jalil Benyacoub, Timo M Buetler, Hans Smola, Stephanie Blum

Nestlé Research Center, Vers-chez-les-Blanc, PO Box 44, CH-1000, Lausanne 26, Switzerland

accepté le 16 Février 2006

Skin is the largest organ of the body and is constantly exposed to physical, chemical, bacterial and fungal challenges. Cutaneous immune surveillance is required to protect the organism from infections but also to detect and remove transformed cells, which eventually may give rise to skin carcinomas [1-3]. On a population basis there is evidence that UV-irradiation alters the immune system and that this constitutes a risk factor for skin tumor development and progression in humans [4]. Tumor transplantation experiments in animal models have clearly demonstrated progressive tumor growth in animals subjected to sub-carcinogenic doses of UV-radiation [5].Indeed, in line with the persistent health concerns over exposure to UV radiation (UVR), research has provided evidence that UV exposure may negatively affect a variety of immune functions and responses both locally and systemically. At the cutaneous level, this effect of UVR has been demonstrated by inhibition of contact hypersensitivity (CHS) reactions following allergen application to areas of UV exposed skin [4, 6]. Apart from this alteration, UVR also induces cutaneous inflammation with development of erythema, edema and hyper-proliferation of the epidermis giving rise to flaking or scaling (reviewed by Soter [7]).Numerous mechanisms are known to be involved in the effect of UVR on the immune system (reviewed by Aubin [3, 8]). UV-induced DNA alterations activate genes coding for immunosuppressive factors, such as IL-10 which is known to modulate the cutaneous immune system [9, 10]. Furthermore, changes in the number and morphology of Langerhans cells (LCs) and their antigen-presenting function have also been reported [4, 11-13].UVR elicits a marked increase in the production of several cytokines that mediate the successive cutaneous recruitment of several types of immune cells: (a) CD11b⁺, CD15⁺ neutrophils which infiltrate the dermis, then the epidermis in the days following UV exposure and play an important role in UVR-induced immunosuppression [14], (b) CD1a^–, CD36⁺, CD11b⁺ macrophage-like cells colonize the dermis, then the epidermis, and, in the days following UV exposure, are reported to be the major source of IL-10 in the skin [13, 15], (c) CD4⁺ memory T-cells appear in the dermis 2 to 4 days post UV-exposure and later in the epidermis promoted by the presence of E-selectin on cutaneous endothelial cells that promote T-cell homing [14]. The effect of UVR on the immune system is thus related to multiple and complex mechanisms.Nutritional intervention, particularly with dietary antioxidants like polyphenols, fatty acids like α-linolenic acid, and vitamins, have been proposed to protect against UV-induced skin damage and to a certain extent skin cancer occurrence (reviewed by Sies & Stahl [16]). There has also been an increasing interest in recent years in nutritional approaches using live microorganisms, or probiotics. It was speculated that the skin status could benefit from reinforced gut homeostasis [17]. Particular attention was paid to specific species of lactic acid bacteria, including Lactobacilli and Bifidobacteria, that are part of the intestinal microbiota.As defined recently by a joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food, including powdered milk with live lactic acid bacteria (2001), “probiotics are live microorganisms, which, when consumed in adequate amounts, confer a health effect on the host”. It is well documented that the endogenous intestinal microbiota plays a crucial role in immune maturation, gut integrity and defense against pathogens [18-20]. Several lactic acid bacteria strains derived from gut microbiota are used in human nutrition with the aim of positively influencing some disease outcomes, such as infectious diarrhea [21]. Several lines of evidence suggest that some probiotic bacteria can modulate the immune system both at the local and systemic level [19, 21], thereby improving immune defense mechanisms and/or down-regulating immune disorders such as allergies or intestinal inflammation [19, 23, 24]. One example of such benefits is the demonstration that consumption of Lactobacillus GG by mothers and babies significantly reduced the incidence of atopic dermatitis in infants at risk [24].The strain Lactobacillus johnsonii NCC 533, La1 [25] used in the present study was isolated from a healthy adult microbiota. This strain was shown to have a strong anti-pathogenic activity against a wide range of entero-pathogens in vitro, in various animal models as well as in humans [26-30]. Furthermore, La1 was also reported to modulate both innate and adaptive immunity in different in vitro, animal and human studies. More specifically, this strain was shown to modulate cytokine expression by human PBMCs in vitro [31]. Schiffrin et al. [32] showed that ingestion of fermented milk containing La1 enhanced the phagocytic activity in blood cells. Moreover, it was shown that administration of fermented milk containing La1 to human volunteers enhanced the specific immunoglobulin IgA response to a Salmonella typhimurium vaccine [33].It is postulated that this capacity of probiotics to modulate the systemic immune status, including the release of regulatory cytokines, might influence skin homeostasis. Based on this, we investigated whether probiotic bacteria such as La1 have the potential to modulate the effect of UVR on the cutaneous immune system by evaluation of their effect on cutaneous hypersensitivity reactions and epidermal Langerhans cell density as well as systemic IL-10 levels.

Materials and methods

Animals

The study was performed at the “Centre International de Toxicologie” (CIT, Miserey, France). Inbred, pathogen-free, female hairless albino Skh:hr1 mice (Charles River, St Aubin-les-Elboeuf, France) aged 8-10 weeks at the initiation of the experiments were used. The animals were maintained in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care International, in accordance with current regulations of the French Health and Human Services. The mice were housed individually under pathogen-free conditions with free access to food (standard laboratory mouse pellets, A.OO4C, UAR, Epinay-sur-Orge, France) and water.

Treatments and oral supplementation

Animals were assigned randomly to twelve groups of 10 mice each. Four groups of mice were used for one product tested as outlined in table 1( Table 1 ).

Lactobacillus johnsonii strain La1 was from the Nestec culture collection (NCC533), hereafter named La1. Bacteria were grown in MRS broth (Difco, Detroit, MI, USA) at 37°C for 16 h. The number of bacteria was determined by colony plating. The bacterial suspension was aliquoted and frozen at – 80 °C. Concentration of viable bacteria after thawing was assessed by plate counts. Each day a vial was thawed and 100 μL of bacterial suspension containing 10⁸ CFU or MRS culture medium (placebo), were administered by gavage to each mouse.

Animals started receiving the respective products 10 days before UV exposure and gavage continued until day 12 (day of DNFB challenge, ( figure 1 )). Thus, the products were fed to the animals for 23 days in total.

At the end of the study, blood was collected from the dorsal artery for IL-10 determination. In addition, 3 mm skin punch biopsies were taken from the back for Langerhans cell determination.
Table 1 Allocation of animals according to supplementation

Product	No UV		2.5 MED UV
Sensitization	Acetone	DNFB	Acetone	DNFB
Control (no product)	Group 1	Group 2	Group 3	Group 4
MRS culture medium	Group 5	Group 6	Group 7	Group 8
La1 10⁸ cfu/day	Group 9	Group 10	Group 11	Group 12

UV radiation source and irradiation procedure

A 1000 W xenon arc lamp including a dichroic mirror (LOT Oriel, Palaiseau, France), equipped with an atmospheric attenuation filter N°81017 (Shott, Clichy, France) and a visible/infrared bandpass blocking filter N°81019 (Shott) was used. This source provided a UVR spectrum simulating solar light (290-400 nm) virtually devoid of any visible and infrared radiation. Irradiance was routinely measured before each exposure session with a Centra ARCC 1600 radiometer (Osram, Berlin, Germany). The integrated irradiance, measured before the beginning of the experiments with a calibrated Bentham DM150 double monochromator spectroradiometer (Bentham, Reading UK), was 1.96 mW/cm² for UVB (290-320 nm) and 14.32 mW/cm² for UVA (320-400 nm) at the skin level.

For UV exposure the mice were lightly anesthetized with a mix of isoflurane/oxygen. A black masking template containing a rectangular opening of 3 × 1.5 cm was placed on their back in order to limit irradiation to the exposure site and to ensure that all mice received comparable UV doses.

Measurement of Skh/hr1 inflammatory response

On day 1 post irradiation with a single solar simulated radiation (SSR) exposure of 2.5 MED, the inflammatory response was determined by assessing erythema and edema ( (figure 1) ). Erythema was evaluated by visual scoring and graded using a pre-established standard scale [34] ranging from 0 (no erythema) to 5 (very intense erythema with strong edema) where a value of 1 corresponds to one minimal erythema dose (MED). This MED was the dose that produced a uniform pale pink color with clearly defined borders 24h post irradiation after a single SSR exposure. This dose was determined previously in 15 Skh:hr1 mice to be 2.68 J/cm² (total SSR spectrum) using a standard protocol (data not shown).

In addition, edema was assessed by measuring the double skin-fold thickness of the dorsal skin with a spring-load micrometer (J15 Käfler, Blet, France).

Contact sensitization protocol

Five days after UV exposure, non-irradiated ventral skin was treated with either 50 μL acetone (control) or sensitized with 50 μL of 0.3% dinitrofluorobenzene (DNFB, Sigma-Aldrich, Lyon, F) in acetone ( (figure 1) ). This sensitization was repeated on the following 2 days. Five days after the last sensitization (12 days after UV exposure), 5 μL of 0.2% DNFB in acetone was applied to the right ear pinna while 5 μL acetone were applied to the left ear pinna of each mouse. After 24 h, ear thickness of both ears was measured under light anesthesia using a micrometer (J15, Käfler). The control groups 1 and 2 (table 1) establish the minimal inflammatory and maximal ear-swelling responses, respectively.

DNFB elicited ear-swelling responses were determined by subtracting the thickness of the acetone-treated from that of the DNFB-challenged ear. Data are presented as the mean ± standard error of the mean (SEM).

Immunoperoxidase staining of epidermal Langerhans cells

The Ia staining of Langerhans cells was done as described previously [35]. Briefly, epidermal sheets from the backs of all UV-exposed and control mice were obtained by incubating skin biopsy specimens in 20 mM ethylenediamine tetracetic acid (EDTA) in phosphate-buffered saline (PBS) pH 7.3 at 37 °C for 120 min. The sheets were then stained with a rat monoclonal antibody (M5/114, American Type Culture Collection, Rockville, MD) raised against mouse Ia [36]. A biotinylated-conjugated rabbit anti-rat IgG (Sigma-Aldrich, Lyon, F) diluted 1/200 in PBS was used as secondary antibody followed by a streptavidin-ABC/HRP complex (Sigma-Aldrich, Lyon, F). Acetyl-ethyl-carbazole developing substrate (Sigma-Aldrich, Lyon, F) was used to localize tissue bound peroxidase-conjugated secondary antibodies. Stained sections were mounted on a glass microscope slide under a glass cover slip and 4 microscopic fields of each sheet/mouse (surface corresponding to 3 mm²) were analyzed for the red stained Ia⁺ cells using an image analyzer (Leica Quantimeter 570, Rueil Malmaison, F). Data are presented as the number of Ia⁺ cells/mm² of epidermis ± SEM. Supporting photographs were taken to illustrate the data.

IL-10 determination in serum

IL-10 levels were measured on day 13 post-UV in serum of all animals by ELISA using 96-well micrometer plates according to the manufacturer’s instructions (Perspective Diagnostics, Cambridge, MA, USA). The level of IL-10 was calculated using a standard curve obtained with recombinant mouse IL-10 (from 0 to 1000 pg/mL). All determinations were performed in duplicates, the results are expressed as pg/mL ± SEM. The results are the pool of all sensitized and un-sensitized animals receiving the same supplementation ± UV exposure (i.e. group 1 and 2, table 1, n = 20).

Statistical analysis

The Mann-Whitney-Wilcoxon test for non-parametric data was used for comparison between two groups. Baseline equivalence between groups was verified and statistical differences were analyzed by analysis of variance when more than two groups were compared (ANOVA test). When two groups were compared, a Tukey t-test was applied. P-values of < 0.05 were considered statistically significant. The analyses were conducted using the SAS software package (version 8.2) and GraphPad Prism (version 4.03) for the inferential analysis and SPSS (version 11.0) for the descriptive analysis.

Results

Erythema and edema

As expected, UV exposure at 2.5 MED caused erythema at the irradiation site in all UV-exposed animals (data not shown). There was no significant difference between any of the UV exposed groups irrespective of whether they received a supplement or not or whether they were sensitized or not (p = 0.211). UV exposure also significantly increased dorsal skin edema measured as skinfold thickness in all exposed groups (data not shown). There was no difference in skinfold thickness between UV exposed groups irrespective of supplementation or sensitization (p = 0.563).

Contact hypersensitivity reaction

UV-irradiation significantly reduced ear swelling in sensitized mice compared with non-supplemented UVR-exposed control animals ( (figure 2) ). La1 supplementation reduced the UVR-induced inhibition of the CHS reaction with ear swelling reaching 80% of the non-irradiated control animals. In the absence of UV-irradiation, La1 had no statistically significant effect on the CHS reaction. Statistical analysis showed that the non-sensitized groups were not different (p > 0.1) from control animals regardless of whether they were exposed to UV or not.

The unexposed, unsensitized group, with no supplementation (group 1, table 1) and the unexposed but sensitized group, with no supplementation (group 2, table 1) are statistically different (p < 0,001, a) in ( figure 2 )) and establish the CHS reaction. The UV-exposed, sensitized group with no supplementation (group 4, table 1) shows a significant reduction in ear swelling after UV exposure (p < 0.001, b) in ( figure 2 )) compared to the unexposed, sensitized group with no supplementation (group 2, table 1) and establishes the UVR-induced immunosuppression.

The important finding of this study is the fact that there was a significant difference between the group receiving La1 and the control (p < 0.0001, d) in ( figure 2 )). The controls show that there was no statistical difference (p = 0.631) between the three unexposed, sensitized groups (receiving La1 or not) that all show a maximal CHS reaction. When all sensitized and UV exposed groups were compared by ANOVA a significant group effect was observed (p < 0.001). The two UV exposed, sensitized, supplemented groups (8 and 12) were then compared individually to the control group 4 by a Tukey t-test. MRS culture medium was not different from the non-supplemented group (p = 0.936, c) in ( figure 2 )).

Epidermal Langerhans cell density

( Figure 3 ) shows that the dorsal skin of unirradiated mice contained on average 1794 ± 36 cells/mm². There was no supplementation effect on LC density in the absence of UV exposure (not shown). After UV exposure epidermal LCs decreased in the control group (2, table 1) to about 290 ± 36 cells/mm².

The group receiving the MRS culture medium was not different from the control group (p = 0.226). In contrast, the number of LCs was not decreased in mice receiving La1 upon UV-irradiation and was at similar levels as in unirradiated animals receiving La1. Indeed, there was a significant difference between mice receiving La1 compared to control and mice receiving MRS (p < 0.001, b) in ( figure 3 )).

Serum IL-10 levels

Average basal serum IL-10 levels in unsensitized animals in the absence of UV exposure were 89 ± 3.6 pg/mL. The sensitization protocol had no effect on the basal IL-10 levels as the levels in sensitized animals not exposed to UV were 89 ± 2.1 pg/mL. Exposure to solar-simulated UVR increased serum IL-10 levels to 170 ± 11.7 and 165 ± 11.7 pg/mL in un-sensitized and sensitized animals, respectively. Since there was no effect of the sensitization protocol the IL-10 values of sensitized and non-sensitized animals were combined for all supplementations. Thus, 13 days after UV exposure, the average IL-10 serum levels were increased in the control and MRS culture medium-supplemented groups to 167 ± 8.1 and 159 ± 11.0 pg/mL, respectively ( (figure 4) ). However, in La1- supplemented mice only 101 ± 3.1 pg/mL of IL-10 were measured even 13 days after UV exposure. This value was not significantly different from unirradiated control mice but significantly below the levels measured in irradiated control mice not receiving probiotic bacteria.

Discussion

In the present study we evaluated the effect of the probiotic strain L. johnsonii La1 (NCC533) on the modulation of skin inflammation and UVR-induced alterations of the cutaneous and systemic immune system determined by a contact hypersensitivity reaction test, epidermal LC density and systemic IL-10 levels.

As expected, UV exposure resulted in the formation of erythema and edema [7, 34, 37] in all the irradiated animals (data not shown). However, there was no significant La1 supplementation effect irrespective of whether mice were subjected to the sensitization protocol or not. Any La1 supplementation effect on kinetic parameters was not evaluated.

As expected, sensitization with DNFB resulted in swelling of the challenged ear. La1 supplementation had no detectable effect on the basal CHS reaction, suggesting that La1 feeding did not influence the CHS reaction in the absence of UV exposure. Upon UV-irradiation there was a strong decrease in the CHS reaction in the control group, thus validating the predicted effect of UV on the skin immune system [38]. This decrease was also observed in animals receiving MRS culture medium demonstrating that the culture medium alone did not confer any protection. However, the CHS reaction in La1- supplemented mice was at approximately 80% of non-UV irradiated animals, suggesting that La1 may be considered as an immuno-protector.

It was recently shown that the probiotic strain L. casei DN-114001 was able to decrease skin inflammation in a DNFB CHS model [39] thus showing an effect in the absence of any UV challenge. It is interesting to note that in our study the La1-mediated effects were undetectable under non-challenging conditions while counteracting an imbalance of the skin homeostasis after UV challenge. To some extent, this finding highlights specificities between probiotic strains. Our data suggest that La1 contributes mainly to reinforce skin homeostasis rather than boosting the cutaneous immune defense per se.

The effect of UV on the induced immune system, as reflected by an inhibition of CHS reaction might be due, at least in part, to a depletion and/or a lack of function of epidermal LC [11, 12, 40, 41]. In our study we found, in agreement with previously published work [34, 42] that the dorsal skin of unirradiated mice contained on average 1794 ± 36 cells/mm². After UV exposure epidermal LCs decreased by about 85%. While this decrease was still observed 13 days post-UVR in both the control and MRS groups, La1 supplementation afforded a significant protection against this UVR-induced decrease in LC density.

In line with these observations we found that the levels of the immuno-suppressive cytokine IL-10 were significantly increased 13 days after exposure to UVR in non supplemented as well as MRS-supplemented animals. This confirms previously published studies showing that IL-10 serum levels were related to the degree of photo-immunosuppression [10, 11]. In the current study we observed that La1-supplementation maintained or restored IL-10 production to levels equivalent to non-UV exposed conditions, thus confirming its ability to preserve the capability of the organism to respond to immunological challenges.

The mechanism underlying immune modulation by probiotics involves, in part, regulation of the composition and/or metabolic activity of the gut microbiota. In addition, a direct interaction of probiotics with the immune system underneath the gut mucosa has also been considered [43, 44]. Indeed, it has been demonstrated that commensal bacteria are able to translocate across the intestinal barrier and induce both polyclonal and specific immune responses [45, 46]. It is postulated that upon interaction of probiotics bacteria (or their components) with the intestinal epithelium and/or directly with dendritic cells, other immune cells such as B and T lymphocytes may be activated (primed) and could release immune mediators and cytokines. These cytokines, bacterial fractions and primed immune cells may reach the skin via the circulation where they could then modulate the local immune status. The present evidence would suggest that La1 could modulate cutaneous UV-triggered immune reactions via priming the immune system in the gut.

Specifically, it has been shown that La1 is able to translocate from the intestinal lumen to Peyer’s patches as well as mesenteric lymph nodes in germ-free mice mono-colonized with La1 and prime the mucosal immune system as demonstrated by an increase in IgA-secreting B cells [47]. The effects of La1 on the immune system is not mediated by increasing the intestinal permeability to luminal antigens, as demonstrated in a human study [48].

It is known that La1 can secrete bioactive components that were shown to exert anti-pathogenic activity in vitro as well as in vivo [27, 30]. However, no secreted immunogenic compound has been identified so far. In the present study we observed that La1-free spent culture supernatant was ineffective in antagonizing UVR-induced immune alterations. However, γ-irradiated dead bacteria elicited a weak but significant protective effect against UVR-induced alterations in the immune response (data not shown). This warrants further investigation.

Finally, although the mechanism, by which a probiotics, including La1, modulates immune functions is not fully elucidated, our data support the idea that oral La1 administration contributes to maintain skin homeostasis and, thus, protect against UV-induced immune effects [22].

Acknowledgements

The authors would like to thank P. Bogdanowictz, L. Sourisseau, S. Tardif and L. Guizelin for technical assistance.

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