Probiotics are believed to alleviate allergic and inflammatory skin disorders, but their impact on pathogenic effector T cells remains poorly documented. Here we show that oral treatment with the probiotic bacteria L. casei (DN-114 001) alone alleviates antigen-specific skin inflammation mediated by either protein-specific CD4+ T cells or hapten-specific CD8+ T cells. In the model of CD8+ T cell-mediated skin inflammation, which reproduces allergic contact dermatitis in human, inhibition of skin inflammation by L. casei is not due to impaired priming of hapten-specific IFNγ-producing cytolytic CD8+ effector T cells. Alternatively, L. casei treatment reduces the recruitment of CD8+ effector T cells into the skin during the elicitation (i.e. symptomatic) phase of CHS. Inhibition of skin inflammation by L. casei requires MHC class II-restricted CD4+ T cells but not CD1d-restricted NK-T cells. L casei treatment enhanced the frequency of FoxP3+ Treg in the skin and increased the production of IL-10 by CD4+CD25+ regulatory T cells in skin draining lymph nodes of hapten-sensitized mice. These data demonstrate that orally administered L. casei (DN-114 001) efficiently alleviate T cell-mediated skin inflammation without causing immune suppression, via mechanisms that include control of CD8+ effector T cells and involve regulatory CD4+ T cells. L. casei (DN-114 001) may thus represent a probiotic of potential interest for immunomodulation of T cell-mediated allergic skin diseases in human.
Citation: Hacini-Rachinel F, Gheit H, Le Luduec J-B, Dif F, Nancey S, Kaiserlian D (2009) Oral Probiotic Control Skin Inflammation by Acting on Both Effector and Regulatory T Cells. PLoS ONE 4(3): e4903. https://doi.org/10.1371/journal.pone.0004903
Editor: Derya Unutmaz, New York University School of Medicine, United States of America
Received: January 8, 2009; Accepted: February 18, 2009; Published: March 20, 2009
Copyright: © 2009 Hacini-Rachinel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by institutional grant from INSERM (infrastructure charges), and from grant from Danone Research Center (Palaiseau, France). F. Hacini-Rachinel is the recipient of a CIFRE this grant from DANONE. Danone as a private company has no role in study design, data collection or interpretation. The authors have no financial conflict of interest.
Competing interests: The authors have declared that no competing interests exist.
Probiotics are defined by FAO and WHO as live microorganisms which when administered in adequate amounts, confer a health benefit to the host . Orally administered probiotics exhibit widespread effects on gut homeostasis and immunomodulation of both mucosal and systemic immunity. Probiotics may counterweight aggressive enteric commensals in the gut and reinforce the barrier function of the epithelium but can also contribute in the regulation of innate and adaptive immune responses of the host under healthy or pathogenic conditions (reviewed in ). Lactic acid bacteria including bifidobacteria and lactobacilli used as probiotics are commensal bacteria of the gut microbiota that could be used for prevention or treatment of chronic allergic and inflammatory diseases, such as inflammatory bowel disease (IBD)  and atopic dermatitis . Recently, studies in patients with IBD  and in animal models of colitis ,  emphasized that probiotics modulate the outcome and severity of intestinal inflammation. Oral administration of a probiotic cocktail (VSL#3) composed of several lactic acid bacteria reduced the severity of chronic T-cell mediated TNBS induced-colitis in mice by acting via a subset of TGFß-bearing cells in the gut lamina propria . In a recently developed mouse model of colitis mediated by cytotoxic CD8 T cells , we observed that L. casei DN-114 001 exerts a protective effect on the severity of intestinal lesions and enhances the function of mucosal CD4+ FoxP3+ Treg in the colon lamina propria (Hacini-Rachinel et al, submitted).
Besides immunomodulation on mucosal inflammation, probiotic may also regulate systemic immune responses that contribute to allergic and inflammatory skin diseases. Studies in human have documented that atopy including atopic dermatitis is less frequently observed in children from mothers who have been under a probiotic diet during pregnancy and the post natal period . The outcome of probiotic treatment on systemic immune responses involved in chronic inflammatory and allergic diseases is still poorly documented. Contact dermatitis is one of the most common skin allergies in western countries . We previously reported that oral administration of L. casei (strain DN-114 001)-fermented milk reduces T cell-mediated delayed-type contact hypersensitivity (CHS) to the hapten 2-4-dinitrofluorobenzene (DNFB) in normal mice , which reproduces the pathophysiology of allergic contact dermatitis in human. Moreover, a clinical study in human with allergic contact dermatitis to Nickel showed that oral treatment with L. casei-fermented milk reduces the severity of prurit and itching (Danone patent EP 1 838 158 B1). However, the impact and mode of action of probiotics, including L. casei, on immune responses involved in the pathophysiology of allergic diseases remains elusive.
Development of allergic contact dermatitis to Nickel , drugs (i.e. antibiotics) and contact sensitizing chemicals (i.e. food conservative, perfume, dyes)  requires two distinct immunological phases. The asymptomatic sensitization phase (i.e. the afferent phase of CHS), which is induced by topical skin exposure to the hapten, results in the priming of hapten-specific T cells. The symptomatic phase (i.e. the efferent phase of CHS) is elicited upon a novel exposure to the same hapten in sensitized individuals and leads within 24/48 hours to the recruitment of the inflammatory skin infiltrate and induction of skin lesion. We and others have extensively characterized the pathophysiology of the CHS response to DNFB in mice and showed that skin inflammation is mediated by hapten-specific IFNγ-producing Tc1-type CD8+ T cells primed within 5 days in skin draining lymph nodes , , , . These CD8 effectors initiate the inflammatory process by performing Fas/Fas ligand and perforine-mediated apoptosis of keratinocytes , . In vivo priming of CD8 effectors and development of the CHS response are independent of CD4 T cell help and the intensity and resolution of skin inflammation is under control of regulatory CD4 T cells (Treg) , – including CD4+CD25+FoxP3+ Treg . Studies in human with Nickel allergy have confirmed that skin inflammation is mediated by Tc1-type CD8 effectors and regulated by CD4+CD25+ Treg .
In the present study, using experimental models of DTH responses mediated by CD4+ T cells specific to a protein Ag, ovalbumin) and by CD8+ T cells specific to the hapten DNFB, we found that oral administration of live L. casei strain DN-114 001 down-regulate skin inflammation mediated by CD4+ or CD8+ T cells. We demonstrated in the CHS model that L casei does not impair the priming or differentiation of hapten-specific CD8 T cells in lymphoid organs, but reduces the frequency of CD8 T cells recruited into the skin upon elicitation of CHS by hapten challenge. We show that this effect requires CD4+ T cells and is accompanied by an effect of L. casei on the frequency and function of Treg.
Materials and Methods
Female C57Bl/6 (2–4 month old) were purchased from Charles River laboratories (L'Arbresle, France). MHC-class II (Aβ°/°)-deficient mice were kindly provided by D. Mathis and C. Benoist, CD1d°/° by L. VanKaer, CD3ε°/° by M. Malissen and bred as homozygotes (C57Bl/6 back-ground, 9th generation) in our animal facilities. All mice were bred at the institute's animal facilities (Plateau de Biologie Expérimentale de la Souris, Ecole Normale Superieure de Lyon) under specific pathogen-free conditions. All experiments were previously approved by the Animal Care and Use Committee according to governmental guidelines.
L. casei DN-114 001 was grown for 16 h at 37°C under aerobic conditions in neutral Man-Rogosa-Sharpe liquid medium (MRS broth, Difco, France). Bacteria were harvested in the stationary phase of growth and stored at 4°C. Viability of L. casei DN-114 001 was >95% for up to 1 week at 4°C. Just before use, bacteria were washed twice in 0.9% NaCl and adjusted at 108 cfu/ml.
Treatment was started at day –14 before epicutaneous sensitization with DNFB and continued until the end of the experiments (i.e. day 12 after sensitization). Mice (5–7 per group) were fed daily by an intragastric stainless steel feeding tube with 200 µl of either live L. casei DN-114 001 (108 cfu/ml), or sterile 0.9% NaCl (control).
Contact hypersensitivity (CHS) to DNFB
Contact sensitivity to DNFB was induced as described . Briefly, mice were sensitized on day 0 by epicutaneous application onto the shaved abdomen of 25 µl of 0.5% DNFB (Sigma Aldrich, St. Quentin Fallavier, France) diluted in acetone/olive oil (4∶1, vol/vol) (i.e. vehicle). On day 5, mice were ear challenged with 0.15% DNFB applied onto the right ear; the left ear received the vehicle alone. Ear thickness was measured with a micrometer (J15 Blet, Lyon, France) before and each day after challenge. Ear swelling was calculated as the difference of swelling between the right and the left ear (ear swelling = ear thickness after challenge – ear thickness before challenge). Results are expressed in µm. Ear swelling after DNFB challenge in unsensitized mice was routinely below 20 µm.
Delayed Type Hypersensitivity (DTH) to ovalbumin
Mice were sensitized subcutaneously with 50 µg of OVA (grade VII, Sigma, France) in 25 µl of saline emulsified with an equal volume of CFA and injected in both sides of the base of the tail. Seven days later the DTH response was elicited by sc challenge with 200 µg of aggregated OVA injected s.c. into the left hind footpad, while the right hind footpad was injected with saline. Aggregated OVA was prepared by heating a 2% solution of OVA at 70°C for 1 h. After cooling, the precipitate was washed and re-suspended in the original volume of saline. Footpad thickness was measured at 24 and 48 h after challenge using calipers. Footpad swelling was determined by subtracting values given by saline-injected footpads from those of Ag-injected footpads.
Isolation of CD8+ T cells and CD4+ CD25+ T cells
After B cells depletion using columns coated by goat anti-mouse and anti-rat IgG BioMag® beads (Qiagen, Germany), CD8+ T cells from splenic, axillary and inguinal lymph nodes of day 5 hapten-sensitized mice were purified by positive selection using anti-CD8 mAb-coated microbeads and magnetic columns (Miltenyi Biotec, France). CD4+CD25+ T cells were purified by positive selection using anti-CD25 mAb-coated microbeads. The percentage of CD8+ T cells and CD4+CD25+ T cells was routinely >90% and 70% respectively as determined by FACS analysis.
Direct in vivo cytotoxicity assay
In vivo analysis of hapten-specific cytotoxic T cells (CTL) was assessed on day 5 after immunization, as described . Mice were injected i.v. with a mixture of target cells in