Anti-allergic Effects of Canine-derived Lactobacillus animalis in Mice and Healthy Dogs

Anti-allergic Effects of Canine-derived Lactobacillus animalis in Mice and Healthy Dogs | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 3 December 2025 V1 Latest version Share on Anti-allergic Effects of Canine-derived Lactobacillus animalis in Mice and Healthy Dogs Authors : Ibuki Yasuda , Mao Kaneki , Mana Ichikawa , Chiharu Ohira , Masaki Nagane , Masaharu Hisasue , Shiro Takeda , Thamonwan Wanganuttara , Jumpei Uchiyama , Keijiro Mizukami , Masahiro Sakaguchi , and Tomoki Fukuyama 0000-0003-3510-3272 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176477066.60314190/v1 309 views 151 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose: Probiotics are promising preventive and therapeutic agents for allergic diseases. This study aimed to investigate the anti-allergic potential of Ligilactobacillus animalis ( L. animalis ), isolated from the feces of healthy dogs, as a candidate probiotic for managing allergic disorders. Experimental Approach: L. animalis strains were isolated from canine fecal samples. Antigen-presenting cell lines were co-cultured with viable L. animalis (1×10 3 –10 6 CFU/mL) for 24 h, and IL-10 levels in the supernatant were quantified using ELISA. The NF-κB pathway activation was examined by detecting p65 phosphorylation in a murine dendritic cell line following 24 h co-culture with viable L. animalis (1×10 6 CFU/mL) using western blotting. The anti-allergic effects of L. animalis were further evaluated in a mouse model of house dust mite–induced atopic dermatitis (AD) and asthma. Key Results: Co-culture with L. animalis significantly increased the IL-10 production by APCs than in untreated controls. NF-κB activation was confirmed by enhanced p65 phosphorylation following L. animalis exposure. In mouse models of AD and asthma, the oral administration of viable L. animalis reduced allergic symptoms, improved histological outcomes, and suppressed local immune responses remarkably. Heat-killed L. animalis also exhibited protective effects, albeit to a lesser extent than the viable bacteria. Conclusion and Implications: The anti-allergic effects of L. animalis are mediated, at least in part, through enhanced IL-10 production by APCs via NF-κB pathway activation. The findings supported the potential of L. animalis as a novel probiotic for the prevention and treatment of allergic diseases in humans and companion animals. Anti-allergic Effects of Canine-derived Lactobacillus animalis in Mice and Healthy Dogs Ibuki Yasuda 1☨ , Mao Kaneki 1☨ , Mana Ichikawa 1 , Chiharu Ohira 1 , Masaki Nagane 2,8 , Masaharu Hisasue 3 , Shiro Takeda 4 , Thamonwan Wanganuttara 5 , Jumpei Uchiyama 5 , Keijiro Mizukami 6 , Masahiro Sakaguchi 7 , Tomoki Fukuyama 1,8 ☨, IY, and MO contributed equally to this work and share first authorship. 1 Department of Veterinary Pharmacology, School of Veterinary Medicine, Azabu University, 1-17-71, Fuchinobe, Chuou-ku, Sagamihara, Kanagawa 252-5201, Japan 2 Department of Veterinary Biochemistry, School of Veterinary Medicine, Azabu University, 1-17-71, Fuchinobe, Chuou-ku, Sagamihara, Kanagawa 252-5201, Japan 3 Laboratory of Small Animal Internal Medicine, School of Veterinary Medicine, Azabu University, 1-17-71, Fuchinobe, Chuou-ku, Sagamihara, Kanagawa 252-5201, Japan 4 Laboratory of Food Science, School of Veterinary Medicine, Azabu University, 1-17-71, Fuchinobe, Chuou-ku, Sagamihara, Kanagawa 252-5201, Japan 5 Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsusimanaka, Kita-ku, Okayama, Okayama 700-0082, Japan 6 Laboratory for Genotyping Development, RIKEN Center for Integrative Medical Sciences, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan 7 ITEA Inc. Institute of Tokyo Environmental Allergy, Tokyo, Japan, 1-33-18, Hakusan, Bunkyo-ku, Tokyo 113-0001, Japan 8 Center for Human and Animal Symbiosis Science, Azabu University, 1-17-71, Fuchinobe, Chuou-ku, Sagamihara, Kanagawa 252-5201, Japan Correspondence: Tomoki Fukuyama, E-mail address: [email protected] (T. Fukuyama) Funding: The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan supported part of this work through the Private University Research Branding Project, 2016-2020. Abstract Background and Purpose: Probiotics are promising preventive and therapeutic agents for allergic diseases. This study aimed to investigate the anti-allergic potential of Ligilactobacillus animalis ( L. animalis ), isolated from the feces of healthy dogs, as a candidate probiotic for managing allergic disorders. Experimental Approach: L. animalis strains were isolated from canine fecal samples. Antigen-presenting cell lines were co-cultured with viable L. animalis (1×10³–10⁶ CFU/mL) for 24 h, and IL-10 levels in the supernatant were quantified using ELISA. The NF-κB pathway activation was examined by detecting p65 phosphorylation in a murine dendritic cell line following 24 h co-culture with viable L. animalis (1×10⁶ CFU/mL) using western blotting. The anti-allergic effects of L. animalis were further evaluated in a mouse model of house dust mite–induced atopic dermatitis (AD) and asthma. Key Results: Co-culture with L. animalis significantly increased the IL-10 production by APCs than in untreated controls. NF-κB activation was confirmed by enhanced p65 phosphorylation following L. animalis exposure. In mouse models of AD and asthma, the oral administration of viable L. animalis reduced allergic symptoms, improved histological outcomes, and suppressed local immune responses remarkably. Heat-killed L. animalis also exhibited protective effects, albeit to a lesser extent than the viable bacteria. Conclusion and Implications: The anti-allergic effects of L. animalis are mediated, at least in part, through enhanced IL-10 production by APCs via NF-κB pathway activation. The findings supported the potential of L. animalis as a novel probiotic for the prevention and treatment of allergic diseases in humans and companion animals. Keywords: Ligilactobacillus animalis ; feces of healthy dogs; IL-10; NF-κB; atopic dermatitis; asthma Abbreviations AD, Atopic dermatitis; LAB, Lactic acid bacteria; CADESI-4, Canine Atopic Dermatitis Severity Index Version 4; CFU, colony-forming units INTRODUCTION Atopic dermatitis (AD) is a chronic, relapsing, and pruritic inflammatory skin disease affecting both humans and companion animals. Globally, AD affects approximately 13% of children and 7–10% of adults, resulting in substantially morbidity, impaired quality of life, and socioeconomic burden (Nutten, 2015; Weidinger & Novak, 2016). In dogs, allergic dermatitis is the most prevalent skin disorder, with an estimated 20–30% prevalence 3 . Certain breeds (Shiba Inu and West Highland White Terriers) have a genetic predisposition, often associated with epidermal barrier defects and Th2-skewed immune responses 4 . Thus, canine AD is a clinically important problem that serves as a valuable comparative model for understanding atopic immunopathogenesis across species. Pharmacological therapy is central to AD management in both humans and dogs. Traditional treatments (glucocorticoids, calcineurin inhibitors) offer immediate. Low-cost efficacy, but are limited long-term adverse effects, including cutaneous atrophy, endocrine suppression, and infection risk. Newer molecularly targeted therapies such as Janus kinase (JAK) inhibitors and anti–IL-31 monoclonal antibodies address refractory pruritus and inflammation (Boerngen, Patel, Pittorino, & Toutain, 2025; Gober, Amodie, Mellencamp, & Hillier, 2025; Gonzales, Aleo, Mahabir, Messamore, & Stegemann, 2024). However, they have high cost, limited accessibility, and incomplete symptom resolution (Gedon & Mueller, 2018; Lee et al., 2025). The limitations underscore the urgent need for safe, affordable, and sustainable complementary strategies to prevent and manage allergic diseases. Lactic acid bacteria (LAB) are promising candidates for such complementary approaches. Probiotics, defined as live microorganisms that confer health benefits when administered in adequate amounts, are known to reinforce epithelial barriers, modulate host–microbe interactions, and promote immune tolerance through the induction of IL-10–producing regulatory T cells (Taverniti & Guglielmetti, 2011). In contrast, postbiotics encompass non-viable microbial cells, cellular components, or metabolites that exert beneficial effects on the host (Aguilar-Toala et al., 2021; Salminen, Stahl, Vinderola, & Szajewska, 2020). Postbiotics offer practical advantages, including an improved safety profile, extended shelf life, and industrial scalability while retaining immunomodulatory properties through the recognition of microbe-associated molecular patterns, such as peptidoglycan, lipoteichoic acid, and exopolysaccharides (Tsilingiri & Rescigno, 2013). Both probiotics and postbiotics have been investigated in allergic and inflammatory conditions (Zorzela, Ardestani, McFarland, & Vohra, 2017). However, to the best of our knowledge, evidence remains fragmented, strain-specific, and inconsistent, and direct head-to-head comparisons between the viable and inactivated formats remain limited. The “healthy-enriched isolate–to–intervention” strategy is an emerging therapeutic concept: isolating bacterial strains enriched in healthy individuals. This approach is justified by the potent, disproportionate immunological effects of even low-abundance microbes. For instance, topical application of Staphylococcus hominis A9, a healthy skin commensal, reduced S. aureus colonization and ameliorated AD lesions (Nakatsuji et al., 2017). Similarly, Roseomonas mucosa derived from healthy skin demonstrated clinical improvement in adult patients with AD by modulating cutaneous inflammation (Myles et al., 2018). The intravaginal administration of Lactobacillus crispatus CTV-05 (LACTIN-V) substantially decreased the recurrence of bacterial vaginosis and urinary tract infections (Cohen et al., 2020; Stapleton et al., 2011). These examples confirm the translational potential of using host-enriched bacteria as therapeutic interventions. Based on this rationale, LAB isolated from healthy dogs are novel candidates for preventing or mitigating allergic dermatitis. Although a minor fraction of the adult canine gut, LAB’s capacity to produce bioactive metabolites and modulate mucosal immunity suggests a functional relevance disproportionate to their low abundance (Mizukami et al., 2019; Uchiyama et al., 2022). However, conventional 16S rRNA-based surveys have not consistently identified LAB as enriched in canine AD cohorts, leaving their contribution to immune homeostasis unclear. Moreover, canine-derived LAB have not been systematically investigated for their anti-allergic properties, nor has the comparative efficacy of viable (probiotic) versus inactivated (postbiotic) preparations been clarified. Epidemiological studies further suggest that early-life exposure to dogs reduces allergic disease risk in children, potentially through shared microbial exposures (Fall et al., 2015; Hesselmar, Aberg, Aberg, Eriksson, & Bjorksten, 1999; Ojwang et al., 2020; Ownby, Johnson, & Peterson, 2002). Supporting this, exposure to dog-associated house dust protected mice against allergen-induced airway inflammation, with enrichment of Lactobacillus johnsonii in protected animals (Fujimura et al., 2014). Whether such protective strains originate from the canine microbiome remains unclear. To address these gaps, we investigated whether LAB enriched in healthy dogs exhibit functional immunomodulatory and anti-allergic activity. We isolated LAB from healthy and AD-affected dogs and evaluated both viable (probiotic) and heat-killed (postbiotic) preparations using in vitro antigen-presenting cell (APC) assays, murine models of AD and asthma, and an exploratory canine model to assess their translational potential for veterinary and human medicine. METHODS Fecal samples from dogs Fecal samples were collected from dogs under anaerobic conditions in sterile polypropylene tubes with filter caps using the AnaeroPack Kenki system (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan) and transported on ice to the laboratory on the same day. Upon arrival, samples were immediately subjected to bacterial isolation. Severity of canine AD was assessed using the Canine Atopic Dermatitis Severity Index Version 4 (CADESI-4). It was evaluated by a veterinarian, and a CADESI-4 score of 10 or higher identified AD. Healthy dogs were defined as having a CADESI-4 score of 10 or lower. Two colonies of Shiba Inu and Shih Tzu dogs, bred and maintained by different owners, were used in this study. Dogs aged less than 5 years were excluded, and finally, 18 dogs were included in the study (Table S1). Animal ethics All animal procedures were conducted in accordance with the Azabu University Animal Experiment Guidelines. Ethical approval was obtained from the Animal Experiment Ethics Committee of Azabu University (Approval No.: 181218-1, 190311-1, and 240313-13). Informed consent was obtained from the dog owners before sample collection. Bacterial isolation Fecal samples were spread on De Man–Rogosa–Sharpe (MRS) culture medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and 5–10 colonies were isolated. Bacteria were purified thrice, and cultured at 37 °C for 24 h under anaerobic conditions using the AnaeroPack Kenki system (Mitsubishi Gas Chemical Co., Tokyo, Japan). PCR was performed using a bacterial 16S rDNA PCR kit (Takara Bio, Shiga, Japan), followed by Sanger sequencing. Bacteria were identified using NCBI Blastn based on 16S rRNA gene sequences. The isolation rates (isolates per dog) were calculated for each species by dividing the total number of isolates by the number of animals (AD, n = 4; healthy, n = 14). A pseudo-count of 0.01 was added to avoid zero counts. Group differences were tested using a two-sample Poisson’s ratio test (score method), and p-values were adjusted for multiple comparisons using the Benjamini–Hochberg procedure. Species with q < 0.1 were considered significant. Bacterial culture and preparation The isolates were cultured in MRS broth (Becton, Dickinson and Company) at 37 °C for 24 h. If required, viable bacterial counts in colony-forming units (CFU) were determined by plating on MRS agar (Becton, Dickinson, and Company). For cell culture and in-vivo experiments, the four strains (L11-2, L13-1, L41-1, and M08-1) were combined in equal proportions to generate a mixed bacterial preparation at the desired concentrations. Genome sequencing, assembly, and analysis Genomic DNA was extracted from bacterial isolates and subjected to whole-genome sequencing at Macrogen (Tokyo, Japan). For strain L11-2, a hybrid sequencing strategy was applied using the PacBio RS II platform for long reads, and the Illumina HiSeq platform for short paired-end reads (2 × 151 bp). PacBio long reads were assembled de novo using HGAP3 (SMRT Analysis v2.3), and the assembly was polished with Illumina reads using Pilon v1.22. For the other isolates (L13-1, L41-1, and M08-1), sequencing was performed using the Illumina HiSeq platform (2 × 151 bp paired-end), followed by library preparation using the TruSeq DNA PCR-Free kit (Illumina). Raw reads were processed to remove the adapter sequences and low-quality bases using Trimmomatic v0.36, and de-novo assembly was performed using SPAdes v3.13.0. Assembly completeness was evaluated using BUSCO v3.0.2, and the bacteria_odb9 dataset, and sequence quality metrics (GC content, Q20/Q30) were obtained using FastQC v0.11.8. Genome annotation was performed using Dfast v1.3.6. Pan-genome analysis was performed with Roary v3.13.0 to generate a core gene alignment of all isolates with other genomes registered in the database. Genomic data were downloaded from the GenBank database (accessed on 15 th Sep, 2025; Table S2). A maximum-likelihood phylogenetic tree was constructed using the FastTree software. The final tree was visualized using iTOL v7. Acquired antimicrobial resistance (AMR) genes were screened using AMRFinderPlus v4.0.23 (reporting thresholds: identity ≥ 90%, coverage ≥ 80%). Virulence-associated genes were surveyed with ABRicate v1.0.1 against VFDB (set A) with ≥ 30% identity and ≥ 70% coverage. Proteins involved in biogenic amine production, including HdcA/B, TdcA, Odc, and AguA/B (Table S3), were searched against the protein sequences (annotated from the genome sequences as described above) using Blastp with BLAST v2.12.0 (identity ≥ 80%, coverage ≥ 80%). The genome sequences were deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers AP045074 (L11-2), BAAIYO010000001-BAAIYO010000031 (L13-1), BAAIYP010000001-BAAIYP010000024 (L41-1), and BAAIYQ010000001-BAAIYQ010000031 (M08-1). Cell lines The murine dendritic cell line DC2.4, murine macrophage cell line RAW264.7, canine macrophage cell line DH82, and human monocyte cell line THP-1 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). DC2.4 and THP-1 cells were maintained in RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France) and penicillin–streptomycin (FUJIFILM Wako Pure Chemical Corporation). DH82 cells were cultured in Eagle’s Minimum Essential Medium (EMEM; FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% FBS and penicillin–streptomycin. RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% FBS and penicillin–streptomycin. All cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Cell stimulation and cytokine measurement Cells were seeded in 96-well plates at a density of 1×10⁵ cells/100 μL, when cultures reached 70–80% confluence. Cells were then exposed to viable L. animalis at various concentrations (1×10⁴–1×10⁸ CFU/mL) for 24 h. For differentiation, THP-1 cells were seeded at 3×10⁶ cells/100 μL in 96-well plates and stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL; Sigma-Aldrich, Tokyo, Japan) for 24 h. After stimulation, the cells were washed with Hanks’ balanced salt solution (HBSS; FUJIFILM Wako Pure Chemicals Co., Ltd.), resuspended in RPMI-1640 medium, and incubated for an additional 24 h to allow differentiation into macrophage-like cells. Differentiated THP-1 cells were then exposed to L. animalis at the same concentration range for 24 h. Supernatants from DC2.4, RAW264.7, DH82, and THP-1 cultures were collected 24 h after L. animalis exposure. Lipopolysaccharide (LPS, 1 μg/mL; Sigma-Aldrich) was used as a positive control. Levels of interleukin (IL)-10 and tumor necrosis factor (TNF)-α were quantified using ELISA kits (DuoSet ELISA, R&D Systems, Minneapolis, MN, USA). Optical density was measured at 450 nm using a microplate reader (iMark; Bio-Rad Laboratories, Inc., Tokyo, Japan). Gene expression analysis in DC2.4 cells DC2.4 cells (3×10⁵ cells/mL) at 70–80% confluence were seeded into 6-well plates and treated with 1×10⁶ CFU/mL viable L. animalis for 24 h. Total RNA was extracted using the NucleoSpin® RNA kit (TaKaRa Bio Inc., Shiga, Japan) following the manufacturer’s protocol. Next, 120 ng of extracted RNA was reverse transcribed using the PrimeScript™ RT Master Mix (TaKaRa Bio Inc.). Gene expression of TNF-α, SOCS3, and STAT3 was quantified by real-time PCR using gene-specific primers (TaKaRa Bio Inc.), PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, Inc., Kanagawa, Japan), and the CFX Duet Real-Time PCR System (Bio-Rad Laboratories, Inc.). Expression levels were normalized to β-actin, and relative expression was calculated using the 2 −ΔΔCt method. Western blot analysis of p65 and Akt phosphorylation in DC2.4 cells DC2.4 cells (3×10⁵ cells/mL) at 70–80% confluence were seeded into 6-well plates and treated with 1×10⁶ CFU/mL viable L. animalis . Total protein was extracted using the M-PERTM Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. A total of 10 µg of protein per sample was separated by SDS-PAGE and transferred to PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc.). The membranes were incubated with primary antibodies against phospho-p65, total p65 (cat. #8214; Cell Signaling Technology, Inc., Danvers, MA, USA), phospho-Akt (Ser473), total Akt (Cat No. #8200, Cell Signaling Technology), and GAPDH (Cell Signaling Technology), followed by incubation with the appropriate secondary antibodies. Immunoreactive bands were visualized using ImmunoStar® Zeta (FUJIFILM Wako Pure Chemical Corporation) and quantified with the iBright imaging system (Thermo Fisher Scientific, Inc.). Acid tolerance assay of L. animalis The probiotic properties of the isolated lactobacilli were evaluated based on their tolerance to low pH as described previously (Halder, Mandal, Chatterjee, Pal, & Mandal, 2017). Briefly, four strains of L. animalis (1×10⁸ CFU/mL) were cultured in MRS broth adjusted to pH 2.0 using hydrochloric acid and supplemented with 4% pepsin (40 mg/mL, Sigma-Aldrich). The medium was sterilized through a 0.22-μm filter before inoculation. Cultures were incubated at 37 °C for 24 h, and viable bacterial counts were determined at 0, 1, 2, and 3 h using Lactobacillus MRS Agar Medium (Becton, Dickinson, and Company). All experiments were performed with at least three independent replicates. Experimental animals and housing conditions Seven-week-old female BALB/c mice (eight mice per group) were obtained from Japan SLC, Inc. (Shizuoka, Japan) and used to establish an allergic asthma model. Seven-week-old female NC/nga mice (eight mice per group) from the same supplier were used to generate the AD model. A maximum of four mice per cage were housed under controlled conditions (12-h light–dark cycle, temperature 22 ± 3 °C, and humidity 55 ± 15%). The mice were provided with a certified pellet diet and tap water ad libitum . A mouse model of AD A mouse model of AD was generated using a previously established method with slight modifications (Fukuyama et al., 2018) . For sensitization, Dermatophagoides farinae (Der f) extract (ITEA Inc., Tokyo, Japan; 1 μg Derf 1 per application) was topically applied to the clipped and tape-stripped back skin and each pinna twice weekly. For preventive studies, daily oral administration of viable L. animalis (n = 6–8) or PBS vehicle (n = 6–8) was initiated one week prior to Derf sensitization and continued for 2 weeks (first setting) or until the end of the experiment (second setting) (Figure S2A and Figure 3A). The third setting was conducted using heat-killed L. animalis on the same schedule as the second preventive setting. For therapeutic studies, viable L. animalis was orally administered after the development of AD skin lesions (day 21) and continued until the end of the experiment (Figure 5A). A total of 4×10⁸ CFU of L. animalis was administered per mouse in the preventive and therapeutic settings. Killed L. animalis was prepared by autoclaving (121 °C, 20 min) or ultraviolet irradiation of the same number of bacteria. L. animalis and PBS were administered approximately 30 min after Derf application. Progression of AD was monitored weekly by measuring scratching behavior, ear and back skin thickness, transepidermal water loss (TEWL), and clinical scores. Clinical scoring (0–4) was assigned to the ears and back as follows: 0, no symptom; 1, mild; 2, moderate; 3, severe; and 4, extreme (Fukuyama et al., 2018) . The summed scores of the ears and back were used in the analysis. In the second preventive setting, scratching behavior was video-recorded for 60 min immediately after Derf application weekly. Scratching bouts were defined as repeated hind limb strokes in the Derf-treated area. At 24 h after the last Derf challenge, the back skin, pinnae, auricular lymph nodes (LN), and blood were collected from each mouse. Samples were processed or stored for histology, cytokine determination, IgE measurement, and flow cytometry. A mouse model of asthma A mouse model of allergic asthma was generated via intranasal sensitization with 25 μg of Derf extract once weekly for 3 weeks, followed by an intranasal challenge with 5 μg of Derf extract on 3 consecutive days prior to sample collection, as described previously (Ando et al., 2024; Ohira et al., 2023; Ookawara et al., 2021) (see Figure 6A). Viable or heat-killed L. animalis was orally administered daily following the same protocol as that used for AD mouse models. To evaluate respiratory function clinically, blood oxygen saturation (SpO₂) was measured using a pulse oximeter (MouseOx® PLUS, Starr Life Sciences Corp., Oakmont, PA, USA) the day before sacrifice, immediately after the final Derf intranasal challenge. On the day after the last Derf challenge, serum, bronchoalveolar lavage fluid (BALF), hilar lymph nodes, and lung tissue were collected from each mouse for subsequent analysis. BALF collection and analysis BALF was collected and analyzed as described previously (Ando et al., 2023; Matsuzaka et al., 2025; Ohira et al., 2023). BALF cell pellets were used to determine the total number of live cells using the CellDrop Cell Counting System, and to perform differential analysis of neutrophils, eosinophils, and macrophages (10,000 events) using a FACSAria III cell sorter (BD Biosciences) with monoclonal antibodies specific for CCR3, CD11c, CD11b, and Gr-1 (BioLegend Inc., San Diego, CA, USA). Cytokine levels, including IL-1β, IL-33, TSLP, KC, RANTES, and EOTAXIN, in the supernatant of the first BALF fraction were quantified using an ELISA kit (DuoSet ELISA kit, R&D Systems) according to the manufacturer’s instructions. Histopathological assessment of skin and lung samples Lung and skin samples were fixed in 10% neutral buffered formalin, paraffin-embedded, and sectioned at 5 µm thickness. The sections were stained with hematoxylin and eosin (H&E). Bronchial, peribronchial, alveolar, and perivascular inflammatory cell infiltrations and hyperplasia were assessed thereafter. Epidermal and dermal inflammation and hyperplasia were evaluated in the skin tissue. All sections were scored semiquantitatively in a blinded manner using the following scale: 0, within normal limits; 1, mild; 2, moderate; 3, severe. Flow cytometric analysis of LNs Single-cell suspensions were prepared from auricular and hilar LNs, as described previously (Matsuzaka et al., 2025; Ohira et al., 2023) . Total cells were counted using a CellDrop™ Cell Counting System (DeNovix Inc., Wilmington, DE, USA). To prevent nonspecific binding, 1×10⁶ cells were incubated with 1 µg of mouse Fc Block (Miltenyi Biotec K.K., Tokyo, Japan) prior to staining with monoclonal antibodies, including anti-mouse CD3, CD4, CD11b, CD11c, CD19, MHC class II, IgE, and DAPI (Miltenyi Biotec K.K.; Sony Biotechnology Inc., Tokyo, Japan). Regulatory T cells (Tregs) were stained using an Inside Stain Kit (Miltenyi Biotec K.K.) and antibodies against CD4, CD25, and FoxP3 (BioLegend, Inc.). The cells were analyzed using a BD FACSAria III cell sorter (BD Biosciences). Cytokine release assay for LNs Single-cell suspensions from LNs (5×10⁵ cells/well) were incubated with Dynabeads Mouse T-Activator CD3/CD28 (Thermo Fisher Scientific, Inc.) for 24 or 96 h. The levels of IL-4, IL-5, IL-9, IL-13, and IL-17 in the supernatants were measured using an ELISA kit (DuoSet ELISA, R&D Systems) according to the manufacturer’s instructions. Measurement of total and Derf-specific IgE levels in serum Serum levels of total IgE (BD Biosciences) and Derf 1-specific IgE (INDOOR Biotechnologies, Inc., Charlottesville, VA, USA) were quantified using ELISA according to the manufacturer’s protocol. Optical density was measured using a microplate reader (iMark; Bio-Rad Laboratories Inc., Tokyo, Japan). Gene expression in skin and lung tissues The frozen skin and lung samples were homogenized using a bead-beater homogenizer (μT-12, TAITEC Corporation, Saitama, Japan). Total RNA extraction and qPCR analysis were performed as described in section 2.8. The expression levels of β-actin, c-kit, GATA3, IL-2, IL-4, IL-10, IL-13, and IL-33 were determined using specific primers (Takara Bio Inc.) and normalized to β-actin. Immunostimulatory effects of L. animalis in dogs A clinical study was conducted on 20 dogs (aged 3–9 years; see Table S4) in accordance with the Azabu University Animal Care and Use Program (Approval No. 240313-13). All owners provided written informed consent. Dogs were divided into vehicle (n = 10) and killed L. animalis (1×10¹⁰ cells/dog/day, n = 10) groups, and received dry food twice daily for 28–35 days. Blood samples were collected before and after the experiment. Populations of dendritic cells and Tregs were analyzed using monoclonal antibodies (anti-canine CD4, CD11b, CD25, MHC class II, FoxP3, and DAPI) and an Inside Stain Kit (Miltenyi Biotec K.K.). Flow cytometry was performed using a BD FACSAria III cell sorter (50,000 events). Serum IL-10 levels were measured using an ELISA kit (DuoSet ELISA; R&D Systems). Statistical analysis Data are presented as means ± standard error of the mean (SEM). Comparisons between two groups were performed using the Student’s t-test, whereas comparisons across three or more groups were analyzed using one-way or two-way ANOVA, followed by Dunnett’s or Šídák’s multiple comparison test, as appropriate. Categorical data were evaluated using Fisher’s exact test. All statistical analyses were performed using Prism 10 (GraphPad Software, San Diego, CA, USA), with a p-value < 0.05 considered statistically significant unless specified otherwise. RESULTS Isolation of bacteria from dog feces We isolated 30 and 127 bacteria from 4 dogs with AD and 14 healthy dogs, respectively, using MRS media. While Enterococcus avium and E. faecium were enriched in dogs with AD (q = 0.0058 and 0.037, respectively), Ligilactobacillus animalis was enriched in healthy dogs (q = 0.071; significance threshold q < 0.10; Table 1). Notably, 18 L. animalis strains were isolated from healthy dogs, whereas none were isolated from dogs with AD. Therefore, four L. animalis strains (M08, L11, L13, and L41) were selected from the isolates of different dogs, and used in subsequent experiments. Genomes of the four strains were sequenced (Table S5). According to the phylogenetic tree derived from the core genomics (Figure 1), our isolates were located among the L. animalis isolates derived from the human gut. The presence or absence of antimicrobial resistance genes in mobile elements, virulence genes, and biogenic amine genes was examined by screening the genome sequences. First, clpP, cpsI, hasB, hasC, htpB, bsh , and ureG were detected in the isolated strains through virulence gene screening, similar to that in other L. animalis genomes (Table S6). The detected genes were not directly related to pathogenicity. ClpP is an ATP-dependent protease subunit involved in stress tolerance and protein quality control. CpsI, HasB, and HasC participate in capsule polysaccharide biosynthesis, which generally confers protection against environmental stress rather than direct toxicity. HtpB encodes a heat shock chaperonin essential for protein folding. Bsh encodes a bile salt hydrolase that contributes to bile resistance and intestinal survival. Second, no antimicrobial resistance gene was found in the genetic mobile elements such as ICE, IME, conjugative elements, and plasmids (Table S7). Third, no biogenic-amine gene was detected (Table S8). Based on these results, the four L. animalis isolates were considered safe for use. In the following experiments, we used a mixture of the four L. animalis strains in order to compensate for strain-to-strain variability, ensure biological diversity, and minimize bias by increasing the generalizability of the findings. In addition, combining multiple strains could provide functional complementarity, thereby enhancing the robustness of the observed immunomodulatory and antiallergic effects. Viable L. animalis induces overexpression of IL-10 and TNF-α in APCs across species To determine the immunomodulatory potential of viable L. animalis , cytokine production was assessed in the macrophage/monocyte cell lines obtained from mice (RAW264.7), dogs (DH82), and humans (THP-1). Stimulation with viable L. animalis remarkably enhanced the secretion of both TNF-α and IL-10 in a dose-dependent manner across all tested species (Figure 2A–F). Notably, TNF-α production was lower in DH82 cells than in murine and human cells, whereas IL-10 production was the highest in DH82 cells (Figure 2A–F). To further characterize the strain-specific properties, acid tolerance and IL-10 induction were evaluated in DH82 cells. The acid survival rates were approximately 10%, 50%, 10%, and 25% for the strains M08, L11, L13, and L41, respectively (Figure S1A). IL-10 production was higher in strains L11 and L13 than in strains M08 and L41, although the differences were not statistically significant (Figure S1B). Viable L. animalis activates NF-κB and PI3K–Akt signaling, leading to STAT3 and SOCS3 upregulation in dendritic cells To investigate the molecular mechanism underlying the anti-inflammatory effects of L. animalis , the mouse dendritic cell line DC2.4 was stimulated with viable bacteria. The stimulation significantly increased the phosphorylation of both NF-κB p65 and Akt than in unstimulated controls (Figure 2D, E). Consistently, the expression of the downstream targets STAT3 and SOCS3 was significantly upregulated (Figure 2F, G). These signaling events correlated with enhanced production of TNF-α and IL-10 (Figure 2H, I). Oral administration of viable L. animalis prevents the development of AD symptoms in mice The preventive effects of oral L. animalis were first assessed in a 2-week pilot AD model. Back skin thickness was significantly reduced in treated mice (Figure S2A), while AD scores and ear thickness showed a downward but non-significant trend (Figures S2B–E). Histology showed reduced epidermal inflammation, with modest changes in hyperplasia and infiltration (Figure S2A, Table S9). L. animalis also decreased DC, Th, and IgE⁺ B-cell infiltration in auricular LNs (Figures S2G–I), suppressed IL-4, IL-5, and IL-17 production, and lowered total and Der f–specific IgE (Figures S3A–F). Skin IL-4, IL-5, IL-17, IL-33, and TSLP were similarly downregulated (Figures S3G–K). A second experiment with continuous administration (Figure 3A) confirmed these effects. Viable L. animalis significantly reduced AD scores, back skin thickness, TEWL, and histologic severity (Figures 3B–F; Table S10), with notable improvement in folliculitis, epidermal hyperplasia, and inflammatory infiltration (Figure 3E). Scratching behavior was unchanged (Figure 3G). Total and Der f–specific serum IgE were significantly decreased (Figures 3H, 3I). Local immune responses paralleled systemic changes. DCs, Th cells, and IgE⁺ B cells in auricular LNs were significantly reduced (Figures 4A–C), and IL-13 and IL-17 were strongly suppressed, with IL-4 trending lower (Figures 4D–F). In lesional skin, IL-2, IL-4, IL-13, IL-33, c-kit , and gata3 expression was reduced (Figures 4G–L), while mast cell numbers were unchanged (Figure 4M). Notably, Tregs were increased in mesenteric LNs (Figure 4N). Heat-killed L. animalis prevents AD symptoms in mice Since heat-killed lactobacilli are more practical to handle than viable strains, their effects were assessed using the same preventive protocol. Oral administration of heat-killed L. animalis significantly reduced AD scores, back and ear thickness, and TEWL than in the controls (Figure S4A–E). Histological analysis confirmed improvement with reduced folliculitis, epidermal hyperplasia, and inflammatory infiltration (Table S11, Figure S4A). Immune profiling showed a non-significant decreasing trend in DCs, Th cells, IgE + B cells, and IL-17 levels in auricular LNs, whereas IL-4 and IL-13 secretion was significantly suppressed (Figure S4F–K). 3.6. Therapeutic effects of viable L. animalis in established AD To assess the therapeutic potential, viable L. animalis was administered after the onset of AD lesions (Figure 5A). Histological evaluation confirmed the suppression of epidermal and dermal inflammation, particularly in the back skin, although changes in hyperplasia and dermal infiltration were modest (Figure 5B, Table S12). AD scores and ear thickness showed a non-significant decreasing trend while back skin thickness was significantly reduced than that in the controls (Figure 5C–E). Unlike those in preventive settings, systemic immune responses were less affected; serum IgE levels, immune cell infiltration, and cytokine production in auricular LNs did not differ significantly between the groups (Figure 5F–L). 3.7. Oral administration of viable and L. animalis inhibits asthma development in mice To assess whether protection extended beyond AD, a Der f–induced asthma model was used (Figure 6A). Viable oral L. animalis significantly improved SpO₂ compared with asthma controls (Figure 6B). Lung KC, RANTES, IL-1β, IL-33, and TSLP expression was significantly reduced, with eotaxin showing a decreasing trend (Figures 6C–H). Histology confirmed marked reductions in alveolar inflammation and multinucleated giant cells (Figure 6I, Table S13). In BALF, eosinophils, neutrophils, and RANTES were significantly decreased, while eotaxin showed a non-significant reduction (Figures S5A–D); total IgE was unchanged (Figure S5E). In hilar lymph nodes, L. animalis significantly reduced DCs, Th cells, and IgE⁺ B cells (Figures S5F–I) and suppressed IL-4, IL-5, IL-9, and IL-13 (Figures S5J–M), without altering total serum IgE (Figure S5N). Heat-killed L. animalis provided partial benefit. Perivascular inflammation was reduced (Figure S6A, Table S14), but SpO₂ remained unchanged (Figure S6B). BALF eosinophils and hilar LN Th and IgE⁺ B cells were decreased, whereas other asthma-related parameters were unaffected (Figures S6C–L). 3.8. Direct immunomodulatory effects of heat-killed L. animalis L41 in dogs The immunological effects of heat-killed L. animalis L41 were examined in dogs 1 month after oral administration. The treatment significantly increased circulating DCs and Tregs compared to both baseline and controls (Figure S7A, B). Consistently, serum IL-10 levels were significantly elevated in the treated dogs (Figure S7C). DISCUSSION Molecularly targeted therapies, such as JAK inhibitors and monoclonal antibodies, are often limited by adverse effects and high costs. Therefore, safe, effective, and affordable strategies are urgently required to prevent the progression and exacerbation of allergic diseases. Among the non-therapeutic approaches, we focused on Lactobacillus strains isolated from the gut microbiota of healthy dogs, and evaluated L. animalis as a novel probiotic candidate for the management of allergic diseases in both humans and dogs. Building on the concept that healthy (non-AD) dogs harbor beneficial bacteria that are absent in dogs with AD, we first compared the gut microbiota of healthy and AD-affected Shiba Inu dogs, a breed genetically predisposed to AD (Mizukami et al., 2019; Uchiyama et al., 2022). The analysis identified 240 bacterial strains unique to healthy dogs, including Bacteroides , Bifidobacterium , Clostridium , Enterococcus , Erysipelatoclostridium , Escherichia , Fusobacterium , Lactobacillus , Proteus , Staphylococcus , and Streptococcus . Twenty of these strains, representing four Lactobacillus species ( L. animalis , L. johnsonii , L. acidophilus , and Limosilactobacillus reuteri ( L. reuteri ) were selected as candidate beneficial bacteria. Previous studies had demonstrated that oral administration of L. johnsonii , L. acidophilus , and L. reuteri from other sources (e.g., humans) can prevent or ameliorate allergic symptoms. However, information regarding L. animalis is limited (Blanchet-Rethore et al., 2017; Kim, Cho, & Kang, 2024; Yu et al., 2023). Considering the novelty and safety endorsement by the European Food Safety Authority, we investigated the efficacy of L. animalis isolated from the canine microbiome as a potential next-generation probiotic for both humans and dogs (Additives et al., 2021). Initially, we examined the effects of viable L. animalis stimulation on APCs, including macrophages and dendritic cells, which are key regulators of immune responses and known targets of several lactobacilli (Kobatake & Arai, 2025; Taverniti et al., 2025). Our results demonstrated that all tested strains of L. animalis significantly enhanced TNF-α and IL-10 production in human, canine, and murine macrophage and dendritic cell lines. The findings indicated that viable L. animalis activates APCs across multiple species with notable species- and strain-dependent differences in cytokine responses. Among the responses, IL-10 is particularly important, since it is produced by APCs, Tregs, and Bregs and plays a central role in maintaining immune homeostasis (Dhatwalia, Sharma, & Kaur, 2025; Zhu et al., 2025). Previous studies had shown that IL-10 deficiency remarkably increases the risk of autoimmune diseases, allergies, and diabetes (Das et al., 2024; Lyu et al., 2023; Nelson et al., 2021; Vetter et al., 2024). Therefore, the significant enhancement in IL-10 production by L. animalis suggested its potential to modulate immune responses in allergic or autoimmune conditions. To elucidate the mechanisms underlying IL-10 induction, we examined the upstream signaling pathways associated with its regulation. In murine dendritic DC2.4 cells, L. animalis stimulation significantly increased the phosphorylation of NF-κB p65 and PI3K–Akt, accompanied by the upregulation of downstream genes STAT3 and SOCS3. This suggested that viable L. animalis can promote an anti-inflammatory phenotype in dendritic cells through the activation of NF-κB and PI3K–Akt pathways, leading to STAT3/SOCS3 induction and enhanced IL-10 production. Based on the in-vitro findings, the immunomodulatory effects of viable L. animalis were further validated in vivo using mouse models of AD. Short-term preventive administration significantly modulated immune cell populations, including DCs, Th cells, and IgE + B cells, and suppressed the production of related inflammatory cytokines; however, its effects on AD symptoms and skin inflammation were modest. To assess the effects of prolonged treatment, continuous administration of viable L. animalis was examined in the same AD model. In this setting, L. animalis inhibited the development of AD symptoms and skin inflammation remarkably, accompanied by the regulation of local immune responses in the auricular LNs and skin. Notably, the proportion of Tregs in the mesenteric LNs was significantly increased by L. animalis treatment. Since Tregs are recognized targets of lactobacilli and a major source of IL-10 (Liu, Fatheree, Dingle, Tran, & Rhoads, 2013; Wang et al., 2022), the finding highlighted an additional mechanism of immune regulation. Consistently, our clinical study on dogs demonstrated that one month of oral administration of heat-killed L. animalis significantly increased the circulating DCs, Tregs, and serum IL-10 levels. Collectively, oral L. animalis administration was found to prevent AD development by suppressing systemic and local allergic responses while enhancing regulatory T cell activity. In contrast, L. animalis treatment had no effect on itching behavior or mast cell infiltration into the skin during AD development. Pruritus in AD is not solely triggered by allergens but is also mediated by cytokines, such as IL-4, IL-13, IL-31, and TSLP (Gonzalez, Bilik, Burke, Pastar, & Yosipovitch, 2025; Lee et al., 2025; Sato, Obonai, Iwata, Ito, & Imafuku, 2025). The itch–inflammation cycle contributes to the disruption of the cutaneous barrier and progression to chronic lesions. Therefore, the lack of an effect on itch behavior represents one of the limitations of oral L. animalis treatment for future clinical applications. Since AD is a multifactorial skin disorder, therapeutic and symptom management strategies are often more clinically relevant than preventive approaches. To evaluate its therapeutic potential, viable L. animalis was administered after the onset of AD lesions. While AD scores and ear thickness showed a non-significant decreasing trend, back skin thickness was significantly reduced than in controls. Unlike in the preventive setting, systemic immune responses were minimally affected, with serum IgE levels, immune cell infiltration, and cytokine production in auricular LNs remaining largely unchanged. The findings indicated that viable L. animalis can provide partial therapeutic benefits for established AD, its efficacy being more pronounced in preventive settings. Heat-killed lactobacilli are easier to handle than viable strains, although their efficacy varies by species and strain (Feng et al., 2024; Santoro, Fagman, Zhang, & Fahong, 2021). Therefore, the effects of heat-killed L. animalis were evaluated using the same protocol. Oral administration significantly reduced AD scores, back and ear thickness, and TEWL than in controls. Immune profiling showed a non-significant decrease in DCs, Th cells, IgE + B cells, and IL-17, whereas IL-4 and IL-13 were significantly suppressed. Thus, heat-killed L. animalis effectively prevented AD, albeit with a narrower immunomodulatory effect than viable bacteria. Next, we examined whether the benefits extended to allergic airway inflammation using a mouse model of asthma. Increasing evidence shows that allergen exposure through the epidermis can initiate systemic allergies and predispose individuals to AD, allergic rhinitis, and asthma (Spergel, 2010). Asthma pathogenesis involves dysregulated immune responses, particularly activation of Th2 cells, B cells, and APCs (Jin, Zhu, Jing, Zeng, & Yan, 2025). To test whether L. animalis could modulate these processes, we evaluated its effects in a Derf-induced asthma model. Viable oral L. animalis significantly improved SpO₂, reduced lung inflammation, and downregulated multiple pro-inflammatory cytokines and chemokines. In BALF, eosinophils, neutrophils, and RANTES were significantly decreased, whereas total IgE remained unchanged. In hilar LNs, viable L. animalis suppressed allergic activation by reducing APC and Th cell populations and limiting Th2/Th17 cytokine secretion, whereas serum IgE levels were unaffected. Heat-killed L. animalis provided partial protection, improved histopathology, and reduced BALF eosinophils and LN Th cells while having minimal effects on SpO₂ and cytokines. Taken together, the findings demonstrated that both viable and heat-killed L. animalis attenuated allergic asthma, with viable bacteria providing broader protective effects. Finally, the immunomodulatory efficacy of heat-killed L. animalis was evaluated in healthy dogs. One month of oral administration significantly increased the levels of circulating DCs, Tregs, and serum IL-10. This suggested that heat-killed L. animalis enhanced the regulatory immune responses in dogs, supporting its potential as an anti-allergic probiotic. Although several clinical studies in dogs with AD have reported the efficacy of lactobacilli, they were largely human- or plant-derived strains and focused primarily on skin lesions (Cauquil & Olivry, 2025; Marsella, 2009; Ohshima-Terada, Higuchi, Kumagai, Hagihara, & Nagata, 2015; Santoro et al., 2021). Our findings highlighted, for the first time, the direct immunomodulatory mechanism of canine-derived L. animalis , which represents a novel and clinically applicable probiotic strategy in veterinary medicine. Despite the promising results, this study had several limitations. The responses in mice and healthy dogs may not fully translate to humans or dogs with established allergic diseases. Heat-killed L. animalis exhibited narrower effects than the viable strains, limiting their therapeutic potential. The therapeutic benefits of viable L. animalis in established AD are partial, particularly regarding pruritus and systemic immunomodulation. Mechanistic exploration was limited to NF-κB and PI3K–Akt pathways, leaving other regulatory pathways unexamined. Additionally, the canine study involved healthy dogs, and the long-term safety, optimal dosing, and efficacy in diseased dogs would require further investigation. In summary, across in vitro , murine, and canine models, L. animalis demonstrated robust immunomodulatory and anti-allergic properties. Viable strains induced TNF-α and IL-10 via NF-κB and PI3K–Akt signaling, suppressed Th cytokines, expanded Tregs, and prevented AD and asthma. Heat-killed strains conferred partial but clinically relevant benefits, particularly for enhancing regulatory immune responses. The findings highlighted canine-derived L. animalis as a promising preventive probiotic with translational potential in veterinary and human medicine. ACKNOWLEDGEMENTS The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan supported part of this work through the Private University Research Branding Project, 2016-2020. We would like to thank Ms. Chiharu Hayakawa, Ms. Sawa Ishihara, Dr. Hironobu Murakami, Dr. Takafumi Osumi, Dr. Sakurako Neo, Dr. Hirotaka Igarashi, and Shoichiro Miyatake for their experimental assistance. We also thank Editage (www.editage.jp) for English language editing. Declaration of Interests M.S., J.U., and T.F. are inventors on a patent application related to the findings described in this manuscript. M.S. is also listed as an inventor on a patent assigned to the Institute of Tokyo Environmental Allergy (ITEA Inc. Tokyo, Japan) related to the findings described in this paper. The remaining authors declare no competing interests. DATA AVAILABILITY STATEMENT The data supporting the findings of this study are available from the corresponding author upon reasonable request. Some data may not be available due to privacy or ethical restrictions. Declaration of AI and AI-assisted technologies in the writing process During the preparation of this work, the authors used ChatGPT to review and refine the text. All content generated with the assistance of ChatGPT was carefully reviewed and edited by the authors, who take full responsibility for the published article. Author contributions T.F. and M.S.: Conceptualization, Data Curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing - Original draft, and Writing - Review & Editing. I.Y., and M.K.: Conceptualization, Data Curation, Formal analysis, Investigation, Methodology, Resources, Supervision, Validation, Writing - Original draft, and Writing - Review & Editing. M.I., C.O., M.N., M.H., S.T., T.W., J.U., K.M., and Y.T.: Investigation, Methodology, Writing - Review & Editing. References Additives, E. 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Country and isolation source are shown in different colors on the right-hand side; the colors enclosed by black lines, are described at the bottom. L. animalis , Ligilactobacillus animalis . Figure 2. Cytokine release and enhancement of NF-κB p65 and PI3K-Akt signaling induced by viable L. animalis in APC lines from different species. TNF-α and IL-10 production in response to viable L. animalis was measured in (A) mouse RAW264.7 cells, (B) canine DH82 cells, and (C) PMA-stimulated human THP-1 cells. DC2.4 cells were co-cultured with viable L. animalis for 24 h. Phosphorylation levels of (D) NF-κB p65 and (E) PI3K-Akt were significantly increased following stimulation. Viable L. animalis treatment also upregulated the gene expression of (F) STAT3 and (G) SOCS3. Correspondingly, the secretion of (H) TNF-α and (I) IL-10 was significantly enhanced. Data are presented as means±SEM (n = 4–8 per group). Statistical significance was determined using Dunnett’s multiple comparison test ( p < 0.05 vs. untreated control). CFU, colony-forming unit; IL, interleukin; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor; L. animalis , Ligilactobacillus animalis . Figure 3. Preventive effects of the oral administration of viable L. animalis in a mouse model of AD. (A) Experimental design for AD induction and preventive treatment with viable L. animalis . (B) Representative back skin images taken 5 weeks after initial Derf treatment in AD control and L. animalis groups. (C) Weekly monitoring of AD scores and (D) back skin thickness (mm). (E) Representative histological images of ear and back skin in each group (scale bar = 100 μm). (F) Cutaneous barrier function assessed by transepidermal water loss (TEWL, g/h/m²). (G) Scratching bouts immediately after Derf treatment were monitored weekly. (H) Total serum IgE and (I) Derf-specific IgE levels (pg/mL) were reduced significantly in the L. animalis treatment group. Data are presented as means±SEM (n = 8 per group). Statistical significance was determined using an unpaired t-test ( p < 0.05, p Figure 4. Modulation of AD-related immune responses by preventive oral administration of viable L. animalis in a mouse model of AD. Oral administration of viable L. animalis significantly inhibited the expansion of (A) dendritic cells (DCs), (B) T helper (Th) cells, and (C) IgE⁺ B cells in auricular lymph nodes (LNs). Secretion of T cell–derived cytokines, including (D) IL-4, (E) IL-5, and (F) IL-17, was significantly suppressed in auricular LNs. Gene expression analysis of skin tissue revealed remarkable reductions in (G) IL-2, (H) IL-4, (I) IL-13, (J) IL-33, (K) c-kit, and (L) gata3, whereas (M) the number of mast cells in lesional skin was not significantly altered. (N) The proportion of regulatory T cells (Tregs) was significantly increased in mesenteric LNs, with representative FACS plots shown. Data are presented as means±SEM (n = 7 per group). p < 0.05 (Dunnett’s multiple comparison test) vs. control group. AD, atopic dermatitis; Derf , Dermatophagoides farinae ; FACS, fluorescence-activated cell sorting; TEWL, transepidermal water loss. Figure 5. Therapeutic effects of oral administration of viable L. animalis in a mouse model of AD. (A) Experimental design for AD induction and therapeutic intervention with viable L. animalis . (B) Representative histological images of back skin from each group (scale bar = 100 μm). (C) AD scores and (D) ear thickness showed non-significant decreasing trends, whereas (E) back skin thickness was significantly reduced compared to controls. Serum levels of (F) total and (G) Derf-specific IgE were not affected by treatment. In auricular LNs, infiltration of (H) DCs, (I) Th cells, and (J) IgE⁺ B cells, as well as cytokine production including (K) IL-4 and (L) IL-13, were not significantly altered. Data are presented as means±SEM (n = 8 per group). p < 0.05 (unpaired t -test) vs. control group. Figure 6. Oral administration of viable L. animalis prevents the development of asthma symptoms in mice. (A) Experimental design of oral administration of viable L. animalis in a Derf-induced mouse model of asthma. (B) SpO₂ (%) was significantly decreased in asthma control mice than in untreated mice, and the reduction was restored by L. animalis treatment. Gene expression levels of (C) Eotaxin, (D) KC, (E) RANTES, (F) IL-1β, (G) IL-33, and (H) TSLP in lung tissue. (I) Representative histological images of lung sections from each group (scale bar = 300 μm or 100 μm). Each result is presented as means±SEM, n = 8 per group. p < 0.05 (Dunnett’s multiple comparison test) vs. control group. BALF, bronchoalveolar lavage fluid; KC, keratinocyte chemoattractant: RANTES, regulated on activation, normal T cell expressed and secreted; SpO₂, percutaneous oxygen saturation; TSLP, thymic stromal lymphopoietin. Supplementary Material File (table_1.docx) Download 19.21 KB Information & Authors Information Version history V1 Version 1 03 December 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Ibuki Yasuda Azabu Daigaku View all articles by this author Mao Kaneki Azabu Daigaku View all articles by this author Mana Ichikawa Azabu Daigaku View all articles by this author Chiharu Ohira Azabu Daigaku View all articles by this author Masaki Nagane Azabu Daigaku View all articles by this author Masaharu Hisasue Azabu Daigaku View all articles by this author Shiro Takeda Azabu Daigaku View all articles by this author Thamonwan Wanganuttara Okayama Daigaku Daigakuin Ishiyakugaku Sogo Kenkyuka View all articles by this author Jumpei Uchiyama Okayama Daigaku Daigakuin Ishiyakugaku Sogo Kenkyuka View all articles by this author Keijiro Mizukami RIKEN Noshinkei Kagaku Kenkyu Center View all articles by this author Masahiro Sakaguchi ITEA Inc Institute of Tokyo Environmental Allergy View all articles by this author Tomoki Fukuyama 0000-0003-3510-3272 [email protected] Azabu Daigaku View all articles by this author Metrics & Citations Metrics Article Usage 309 views 151 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Ibuki Yasuda, Mao Kaneki, Mana Ichikawa, et al. 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