Effects of Canine-Derived Bifidobacterium animalis subsp. lactis DS008 Culture Supernatants on In Vitro Canine Keratinocytes

TL;DR

This study shows that postbiotics from a canine-specific Bifidobacterium strain reduce inflammation and improve skin barrier function in dogs.

Contribution

The study demonstrates the anti-inflammatory and skin barrier-enhancing effects of a canine-derived Bifidobacterium postbiotic for the first time.

Findings

DS008 supernatants reduced inflammatory cytokines like TNF-α, IL-13, TSLP, and IL-31 in canine keratinocytes.

Treatment increased keratin 10 expression, supporting skin cell adhesion and barrier formation.

DS008 supernatants restored stratum corneum structure in canine epidermis models.

Abstract

This study investigated the impact of postbiotics derived from Bifidobacterium animalis subsp. Lactis DS008 on in vitro canine keratinocytes. The DS008 supernatants significantly reduced the expression of inflammatory cytokines linked to itching and skin irritation. In addition, treatment with DS008 supernatants significantly increased keratin 10 expression, which helps skin cells adhere to one another and form a protective barrier. When applied to reconstructed canine epidermis models with a disrupted stratum corneum, the DS008 supernatants induced the restoration of the stratum corneum’s structure. Altogether, these results indicate that postbiotics from canine Bifidobacterium animalis subsp. lactis DS008 may represent a promising anti-inflammatory approach and support skin barrier health in dogs.

Microorganisms residing on the skin play a crucial role in maintaining both the integrity of the skin barrier and immune function. This study examined the effects of culture supernatants from canine-derived Bifidobacterium animalis subsp. lactis DS008 on canine keratinocytes in vitro. To induce cytokine production, canine progenitor epidermal keratinocytes (CPEK) were indirectly co-cultured with Malassezia pachydermatis using an insert well system to prevent direct cell-to-yeast contact, and CPEK were supplemented with 0.1%, 1%, and 10% of DS008 supernatants. mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-13, thymic stromal lymphopoietin (TSLP), IL-31, and keratin 10 were analyzed by real-time PCR. The protein concentrations of TSLP and IL-31 were measured by ELISA. Additionally, DS008 supernatants were applied to reconstructed canine epidermis (RCE) models exposed to lipopolysaccharide to evaluate changes in epidermal architecture. Treatment with DS008 supernatants showed significant reductions in mRNA expression of TNF-α, IL-13, TSLP, and IL-31, as well as decreased protein levels of TSLP and IL-31. Furthermore, keratin 10 mRNA expression was significantly increased, and RCE analysis demonstrated that DS008 supernatants ameliorated stratum corneum deformation. These findings suggest that postbiotics derived from canine-derived Bifidobacterium animalis subsp. lactis DS008 is a potential candidate with anti-inflammatory properties.

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1. Introduction

As the largest organ in the body, the skin is colonized by a complex community of microorganisms and plays a crucial role in protecting against invading pathogens and regulating the immune system [1]. The skin microbiome produces a large number of metabolites that play a vital role in developing the skin barrier and reducing the pathogenic microbiome [1]. A recent pyrosequencing study demonstrated that higher microbial richness and diversity are observed in healthy canine skin compared to those of atopic dermatitis, while lower microbiome species are associated with dogs with atopic dermatitis [2,3].

Previous studies in humans have suggested that probiotics have great potential to regulate various skin conditions. Topical formulations containing 5% bacterial lysate (inactivated extract) of the non-pathogenic Gram-negative bacterium Vitreoscilla filiformis significantly decreased the clinical sign index score and pruritus levels in human atopic dermatitis (AD). In addition, V. filiformis lysate reduced Staphylococcus aureus colonization of the skin and significantly decreased transepidermal water loss [4]. Furthermore, decreased skin dryness and increased skin resistance were observed when a topical cream containing Bifidobacterium longum was applied to human skin with reactive skin lesions [5].

Similar to findings in human studies, oral administration of live probiotics of Bifidobacterium longum may help resolve skin lesions in dogs with atopic dermatitis (AD) [6]. A recent study also demonstrated that oral administration of probiotics (Bifidobacterium bifidum, Lactobacillus acidophilus, and Enterococcus faecium) can effectively ameliorate clinical signs of dogs with AD by improving gut microbial dysbiosis [7].

Recent research into microbiome-targeted ingredients for skin health has shifted towards postbiotics—bioactive compounds from probiotic organisms—which, unlike probiotics, contain non-living microbial components and therefore reduce risks associated with live cells. Research in humans has demonstrated that postbiotics contribute to skin health through their anti-inflammatory and antioxidant properties, enhanced skin barrier function, improved hydration, and promotion of beneficial microbes while suppressing pathogenic organisms [8]. In dogs, oral administration of an indole-rich postbiotic reduced pruritus by 27%, suggesting its potential for broader immune-related effects via the gut–skin axis [9]. In addition, oral administration of a postbiotic and probiotic supplement containing Bifidobacterium animalis subsp. lactis increased bacterial diversity, suggesting a promising strategy to improve skin health by modulating the microbiota and reducing the risk of skin infections [10]. However, the mechanisms in canine skin, particularly regarding potential impact on canine keratinocytes, remain poorly understood. The present study aimed to investigate the effects of postbiotics derived from canine-specific B. animalis subsp. lactis DS008 on cytokine expression in canine keratinocytes.

2. Materials and Methods

2.1. Preparation of Bifidobacterium animalis subsp. lactis DS008

This study used a novel microorganism, B. animalis subsp. lactis DS008, isolated from the facial skin of a healthy dog (accession number KCCM12993P; received 28 May 2021). The morphological findings of the strain were observed using scanning electron microscopy (SEM), and genetic identification was performed using 16S rRNA analysis with primers 27F and 1492R for amplification and 27F, 518F, 907R, and 1492R for sequencing, as previously reported [11,12]. Strain DS008 was cultured in tryptic soy broth at 37 °C for 72 h under anaerobic conditions and then centrifuged and filtered through a 0.45 µm filter (CLS431225, Corning, NY, USA). The filtered medium was applied to canine keratinocytes.

2.2. Cell Culture

Canine epidermal keratinocyte progenitors (CPEK) were obtained from CELLnTEC Advanced Cell Systems (Bern, Switzerland). The CPEK cells were cultured in CnT-09 medium (CELLnTEC, Bern, Switzerland) under conditions of 5% CO_2_ at 37 °C in a humidified incubator. For real-time PCR analysis targeting keratin 10 (K10), CPEK were plated in 6-well plates and then treated with 0.1%, 1%, or 10% filtered culture supernatant from strain DS008 for 24 h. Additionally, to induce cytokine expression in canine keratinocytes, CPEK were co-cultured with Malassezia pachydermatis, which has been shown to induce an immune response in keratinocytes [13]. To prevent direct cell-to-yeast contact, CPEK were indirectly co-cultured with Malassezia pachydermatis using an insert well system. The M. pachydermatis strain was sourced from the Korean Collection for Type Cultures (KCTC 27588, Jeongeup-si, Republic of Korea) and grown on modified Dixon agar at 32 °C for 5 days. Yeast cell concentrations were determined by trypan blue exclusion and adjusted to 1 × 10^7^ CFU per insert. M. pachydermatis was then co-cultured with CPEK in the presence of filtered strain DS008 culture supernatant for 8 h.

2.3. RNA Isolation and Real-Time PCR

Total RNA was isolated from CPEK using TRIzol reagent according to the manufacturer’s instructions (TaKaRa, Shiga, Japan). cDNA was synthesized from 1 μg of total RNA using Reverse Transcription Premix (Elpis-biotech, Daejeon, Republic of Korea) under the following reaction conditions: 45 °C for 45 min and 95 °C for 5 min. Gene expression signals were quantified using real-time PCR with StepOne Plus system software V2.3 (ABI 7300, Applied Biosystems, Foster City, CA, USA). Real-time PCR amplification reactions were performed using SYBR Green PCR Master Mix with premixed ROX (Applied Biosystems, Foster City, CA, USA) targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interleukin (IL)-13, tumor necrosis factor-α (TNF-α), thymic stromal lymphopoietin (TSLP), IL-31, and K10. The primer pairs (Bioneer, Daejeon, Republic of Korea) used in this study are summarized in Table 1. The reaction conditions were as follows: initiation at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 1 min. GAPDH expression was used as an internal control. Relative gene expression was calculated using the 2^−ΔΔCt^ method.

2.4. Immunoassay

Canine TSLP and IL-31 levels were quantified using commercially available ELISA kits (Mybiosource, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, CPEK cell culture supernatants were collected and centrifuged at 1000× g for 10 min to remove debris. Samples and standards were added to 96-well plates pre-coated with capture antibodies and incubated at room temperature for the recommended time. After washing, biotinylated detection antibodies were applied, followed by a streptavidin–HRP conjugate. Absorbance was measured at 450 nm using a microplate reader. Cytokine concentrations were calculated from standard curves generated with known concentrations of recombinant TSLP and IL-31.

2.5. Reconstructed Canine Epidermis

Reconstructed canine epidermis (RCE; CleSKIN Canine, CLECELL, Seongnam-si, Republic of Korea), cultured for 12 days under air–liquid interface (ALI) conditions to promote differentiation, was purchased to further investigate the effects of DS800 supernatants. After pre-incubation, samples were divided into vehicle, LPS-treated, or LPS+DS008-treated groups. To induce inflammation in RCE, LPS (10 μg/mL) was added for 72 h, and DS008 culture supernatant (1%) was also added to the media in the LPS+DS008-treated groups. Vehicle samples received only fresh medium. Epidermis samples were fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin before being examined under a Leica DM2500 optical microscope (Leica, Wetzlar, Germany).

2.6. Statistical Analysis

All experiments were repeated at least three times, and each experiment was performed in triplicate. Results are presented as mean ± standard deviation. Statistical analysis was performed using the SPSS statistical package version 25.0 (SPSS Inc., Chicago, IL, USA) with a two-tailed Student’s t-test. p < 0.05 and p < 0.01 were considered statistically significant.

3. Results

3.1. Identification of Bifidobacterium animalis subsp. lactis DS008

To identify bacterial species, SEM and 16S rRNA gene sequencing were performed. SEM analysis of the strain reveals rod-shaped bacteria that frequently form pairs or clusters (Supplementary Figure S1). Preliminary sequence identification, conducted by comparison with the National Center for Biotechnology Information database, indicated that the strain belongs to the species B. animalis subsp. lactis. Therefore, the bacterial species used in this study were identified as B. animalis subsp. lactis.

3.2. Reduction in Inflammatory Cytokines IL-13, TNF-α, and TSLP Following Treatment with DS008 Culture Supernatants

To investigate the effects of DS008 culture supernatants on cytokine expression in canine keratinocytes, CPEK cells were co-cultured with M. pachydermatis, and quantitative real-time PCR was performed. As shown in Figure 1, quantitative real-time PCR demonstrated that co-culture with M. pachydermatis resulted in more than a twofold increase in IL-13 Figure 1A, TNF-α Figure 1B, and TSLP Figure 1C mRNA expression in CPEK cells compared to control samples. In addition, treatment of CPEK cells with 0.1%, 1%, and 10% DS008 culture supernatants significantly attenuated the elevated mRNA expression levels of these cytokines Figure 1A–C. Furthermore, ELISA results indicated that the increased TSLP concentrations induced by M. pachydermatis co-culture were markedly reduced upon supplementation with DS008 culture supernatants Figure 1D. Therefore, DS008 culture supernatants decreased the expression of inflammatory cytokines IL-13, TNF-α, and TSLP.

3.3. Reduction in IL-31, a Pruritogenic Cytokine, Following Treatment with DS008 Culture Supernatants

To further investigate the effects of DS008 culture supernatants on pruritic pathways, a pruritogenic cytokine, IL-31, was analyzed. CPEK co-cultured with M. pachydermatis showed elevated mRNA expressions of the pruritogenic cytokine IL-31, which were significantly reduced by 0.1%, 1%, and 10% DS008 culture supernatants Figure 2A. ELISA also confirmed that DS008 supplementation at these concentrations lowered IL-31 levels induced by M. pachydermatis co-culture Figure 2B. Therefore, DS008 culture supernatants decreased the expression of the pruritogenic cytokine IL-31.

3.4. Treatment with the DS008 Culture Supernatants Restored the SC Structure

To analyze the effects of DS008 culture supernatants on the epidermal barrier, K10 expression, and morphological changes in RCE, tests were performed after adding DS008 culture supernatants. Real-time PCR analysis revealed that K10 mRNA expression increased in CPEK cells supplemented with 1% or 10% DS008 compared to the control Figure 3A. Additionally, in the RCE model, treatment with LPS caused the well-preserved SC structure in the control group to become loosened. However, when RCE was supplemented with 1% DS008 culture supernatant, the SC structure was restored to resemble that of the controls Figure 3B. Therefore, treatment with DS008 culture supernatant alleviated damage to the stratum corneum (SC).

4. Discussion

The cell-free culture supernatant produced by probiotics during fermentation contains beneficial metabolites and components, such as short-chain fatty acids and antimicrobial peptides. It is thus considered a key source of postbiotics [17]. In this study, we investigated the effects of postbiotics on canine skin by examining culture supernatants of canine-derived B. animalis subsp. lactis DS800. DS800 supernatants significantly reduced cytokine expression and increased K10 expression.

Previous studies reported that bacterial cell-free culture supernatants, classified as postbiotics, have beneficial effects on skin regeneration and reducing inflammation. In a previous study, the supernatant of lactic acid bacteria significantly decreased the expression of chemokines (macrophage-derived chemokine and thymus and activation-regulated chemokine) and cytokines (IL-4, IL-5, IL-25, and IL-33) in TNF-α/interferon-γ-induced HaCaT keratinocytes [18]. In addition, similar to the present study, metabolites derived from B. animalis subsp. lactis, including cell-free supernatants, have been shown to modulate CCL5 expression and epidermal barrier-related markers, such as filaggrin and the hyaluronic acid-associated pathway [19]. Consistent with previous findings in human HaCaT keratinocytes, this study suggests that bacterial cell-free culture supernatants may exert anti-inflammatory effects and preserve stratum corneum structure.

TSLP, which plays a crucial role in AD by inducing Th2-type immune responses, is elevated in both lesional and non-lesional skin of dogs with AD [15]. IL-13, another Th2 cytokine, promotes allergic inflammation and IgE production, and elevated plasma levels are observed in dogs with AD [20]. TNF-α is involved early in allergen sensitization and drives the inflammation cascade, with higher concentrations observed in dogs with AD [21]. In this study, culture supernatants from canine-derived B. animalis subsp. lactis reduced mRNA expression of TSLP, IL-13, and TNF-α, as well as TSLP protein concentration, in M. pachydermatis co-culture. These results indicate that supernatants of canine-derived B. animalis subsp. lactis DS800 may downregulate key inflammatory cytokines involved in canine AD.

IL-31 is a pruritogenic cytokine and directly induces itching by binding to IL-31 receptor alpha (IL-31RA) on sensory neurons and immune cells [21]. Dogs with atopic dermatitis exhibit increased IL-31 expression in lesional skin, which correlates with the severity of pruritus and inflammation [21,22]. In addition, blocking IL-31 or IL-31RA by monoclonal antibodies or vaccines significantly reduces pruritus and improves skin condition in dogs with AD [21]. Consequently, the observed decrease in IL-31 expression in this study suggests that culture supernatant from canine-derived B. animalis subsp. lactis DS800 may serve as a potential agent for reducing IL-31-mediated pruritus in dogs.

The present study showed that culture supernatants of a canine-derived B. animalis subsp. lactis increased K10 mRNA expression. K10 is a structural protein that helps maintain epidermal integrity and regulates keratinocyte differentiation. Additionally, it plays a crucial role in forming the skin’s protective barrier by stabilizing the keratinocyte structure [23]. Furthermore, in this study, the disturbed SC structure in RCE was reconstructed using DS800 culture supernatants. Taken together, these results suggest that the culture supernatant of canine-derived B. animalis subsp. lactis DS800 plays a protective role in preserving SC integrity.

In the present study, canine keratinocytes co-cultured with M. pachydermatis produced cytokines TNF-α and TSLP. In vitro cytokine production induced by Malassezia spp. has been reported in human keratinocytes. Malassezia yeasts, including M. furfur and M. globosa, induce TNF-α, IL-6, IL-8, and IL-1α production by human keratinocytes, and cytokine expression differed among the Malassezia yeasts [24]. In addition, M. globosa or M. restricta induced a marked increase in TSLP secretion in human keratinocytes [25]. The effect of M. pachydermatis was also reported in human keratinocytes, which revealed upregulated expressions of IL-1β, TNF-α, IL-6, and IL-8 after co-culture with M. pachydermatis [13]. However, little is known about the effects of M. pachydermatis on cytokine production in canine keratinocytes. In the present study, M. pachydermatis induced the expression of inflammatory cytokines TNF-α, IL-13, TSLP, and IL-31. Therefore, an in vitro system of co-cultured keratinocytes with M. pachydermatis could help investigate inflammatory responses in canine keratinocytes.

The potential limitations of the present study are as follows: (1) The effect of canine-derived B. animalis subsp. lactis DS008 culture supernatants were investigated only in vitro. Future large-scale studies, including in vivo clinical examinations, are needed. (2) Other components of the skin barrier function, such as epidermal lipids and various inflammatory cytokines, were not investigated. Further studies to analyze their impact on a broader range of cytokines, chemokines, and epidermal barrier components are required. (3) The entire culture supernatant of B. animalis subsp. lactis DS008 was used; therefore, it was unclear which substance had beneficial effects on canine keratinocytes. According to previous studies, the culture supernatants of B. animalis subsp. lactis strains are known to produce biologically active metabolites, including short-chain fatty acids, low-molecular-weight peptides, and cell-wall-derived components. These postbiotic substances have been reported to modulate keratinocyte differentiation, enhance epidermal barrier-related protein expression, and attenuate pro-inflammatory signaling [19]. Therefore, future studies performing a comprehensive metabolomic analysis of DS800 supernatant to identify individual bioactive compounds will provide further understanding of the observed effects on cytokine expression and epidermal barrier restoration.

5. Conclusions

As multiple factors contribute to the pathogenesis of canine inflammatory skin diseases such as AD, a multidisciplinary therapeutic approach is needed. Therefore, microorganism-based topical formulations may support the beneficial effects of other medical drugs. This study provides fundamental data on the beneficial effects of postbiotics derived from canine B. animalis on canine keratinocytes.