Application of Lactobacillus paracasei HB89 mitigates aluminum hydroxide combined with OVA allergen in an allergic animal model

Application of Lactobacillus paracasei HB89 mitigates aluminum hydroxide combined with OVA allergen in an allergic animal model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Application of Lactobacillus paracasei HB89 mitigates aluminum hydroxide combined with OVA allergen in an allergic animal model Hao-Chen Lin, Ding-Ying Lai, Sing-Yi Gu, Chia-Yi Tseng, Wei-Chun Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8452034/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Allergic airway inflammation, primarily driven by a Th2-biased immune imbalance, remains a significant global health concern. This study investigated the immunomodulatory potential of Lactobacillus paracasei HB89 in a murine model of ovalbumin (OVA)-induced allergic asthma. Female BALB/c mice were sensitized and challenged with OVA to establish an allergic airway hyperresponsiveness (AHR) model, while L. paracasei HB89 was administered via oral gavage daily for ten consecutive weeks. Results demonstrated that HB89 intervention was well-tolerated and significantly enhanced innate immune surveillance, evidenced by increased natural killer (NK) cell cytotoxicity and phagocytic activity. While maintaining splenic lymphocyte homeostasis, HB89 markedly attenuated Th2-driven systemic responses, as shown by a significant reduction in total serum IgE. Furthermore, HB89 treatment effectively modulated the inflammatory microenvironment by downregulating IL-4 and IL-5 secretion in both stimulated splenocytes and bronchoalveolar lavage fluid (BALF). Crucially, HB89 administration significantly mitigated AHR, markedly reducing methacholine-induced airway resistance. Collectively, these findings suggest that L. paracasei HB89 alleviates allergic airway inflammation by rebalancing Th1/Th2 cytokine profiles and strengthening innate immunity, positioning it as a promising functional probiotic for managing allergic airway diseases. Lactobacillus paracasei HB89 Allergic airway inflammation Th1/Th2 balance Airway hyperresponsiveness Innate immunity Probiotics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Allergic airway inflammation, a hallmark of allergic asthma, is a chronic inflammatory disorder of the airways characterized by reversible airflow obstruction and airway hyperresponsiveness (AHR) [ 1 , 2 ]. Globally, the prevalence of allergic diseases has increased significantly, driven by complex interactions between genetic susceptibility and environmental triggers. The immunopathology of allergic asthma is primarily dictated by a Th2-biased immune response, where the overproduction of cytokines such as interleukin (IL)-4 and IL-5 facilitates immunoglobulin E (IgE) synthesis and eosinophilic infiltration into the lungs [ 3 , 4 , 5 ]. Despite the availability of pharmacological treatments like corticosteroids, concerns regarding long-term side effects have accelerated the search for safer, complementary dietary interventions, particularly probiotics [ 6 , 7 ]. Probiotics, defined as live microorganisms that confer health benefits to the host, have demonstrated significant potential in modulating systemic and mucosal immune responses [8] . Specifically, members of the Lactobacillus genus have been shown to rebalance Th1/Th2 profiles and alleviate allergic symptoms in various murine models [9] . Among these, Lactobacillus paracasei has emerged as a potent immunomodulator. Previous studies have indicated that specific strains of L. paracasei can mitigate particulate matter-induced airway inflammation [10] . However, the systemic impact of L. paracasei HB89 (BCRC910811) on innate immune surveillance and its specific efficacy in an ovalbumin (OVA)-induced classic allergic model—characterized by acute respiratory functional changes—remains to be fully elucidated. In this study, we investigated the therapeutic potential of L. paracasei HB89 in a standardized OVA-sensitized BALB/c mouse model following official health assessment protocols [11] . Beyond evaluating the typical Th1/Th2 cytokine balance and IgE levels, we specifically focused on the enhancement of innate immune functions, including natural killer (NK) cell activity and phagocytic capacity. Furthermore, we utilized the Penh index to objectively quantify the mitigation of AHR. Our findings aim to provide a comprehensive toxicological and physiological basis for HB89 as a functional probiotic candidate for the preventive management of allergic airway diseases. Materials and Methods Animal model Forty female BALB/c mice (8 weeks old) were purchased from Lesco Biotechnology Co., Ltd. (Taiwan). All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Super Laboratory Co., Ltd. (Approval No. 111-14g). Mice were housed in a controlled environment with a 12/12-h light/dark cycle at 22 ± 2°C and 55 ± 15% humidity, with ad libitum access to a standard diet and sterilized water. All animal procedures were conducted in strict accordance with the ARRIVE guidelines and the "Assessment Method for Assisting the Regulation of Allergic Constitution" promulgated by the Taiwan Ministry of Health and Welfare [11] . Preparation and Dosage of L. paracasei HB89 L. paracasei HB89 (BCRC910811) was cultured anaerobically in MRS broth at 37°C. Bacterial cells were harvested via centrifugation (3000 × g, 15 min, 4°C) and resuspended in sterile PBS. The dosage for the experimental groups was determined based on the human equivalent dose (HED). Mice were randomly divided into four groups (n = 10/group): Normal Control : Administered reverse osmosis (RO) water. Untreated Control (OVA-sensitized) : Administered RO water. Low-dose (1X) : Administered HB89 at 0.6150 g/kg BW daily. High-dose (2X) : Administered HB89 at 1.2300 g/kg BW daily. All treatments were administered via oral gavage at a volume of 10 mL/kg BW daily for 72 days. Administration design Group Allocation and Treatment Regimen SPF-grade female BALB/c mice were randomly assigned to four experimental cohorts (n = 10 per group): a normal control group, an ovalbumin (OVA)-sensitized negative control group, and two intervention groups receiving Lactobacillus paracasei HB89 at low (1X) and high (2X) dosages. Both the normal and negative control groups were administered reverse osmosis (RO) water daily via oral gavage. Probiotic dosages were extrapolated from the Human Equivalent Dose (HED) based on metabolic body surface area normalization (Km = 12.3 for mice). Accordingly, the 1X and 2X groups received daily dosages of 0.6150 g/kg and 1.2300 g/kg body weight (BW), respectively. To maintain therapeutic precision and microbial stability, treatment solutions were prepared fresh daily by reconstituting HB89 in RO water to final concentrations of 0.0615 g/mL (1X) and 0.1230 g/mL (2X). All substances were delivered via oral gavage using a 20-gauge feeding needle (70 mm length) at a constant volume of 10 mL/kg BW. The administration was performed daily for 72 consecutive days, spanning the entire duration of the experimental protocol. Establishment of the OVA-Induced Allergic Asthma Model The murine model of allergic asthma was established following the standardized "Assessment Method for Assisting the Regulation of Allergic Constitution" promulgated by the Taiwan Ministry of Health and Welfare. Mice were sensitized via intraperitoneal (i.p.) injections of 0.2 mL sensitization solution containing 2 mg/mL ovalbumin (OVA; Sigma-Aldrich, St. Louis, MO, USA) emulsified with 2 mg of aluminum hydroxide adjuvant (Alum; Sigma-Aldrich) on days 35, 49, and 63 of the experimental period. To induce acute airway inflammation and hyperresponsiveness, mice underwent respiratory challenges via intranasal (i.n.) administration of 2% OVA (50 µL per mouse) on days 70, 71, and 72. The normal control group received an equivalent volume of sterile phosphate-buffered saline (PBS) throughout the sensitization and challenge phases. Euthanasia and subsequent sample collection were performed on day 73, exactly 24 hours following the final respiratory challenge. Testing Items : Daily animal observations were conducted to determine if the test mice exhibited any abnormal symptoms or adverse effects. Abnormal symptoms or deaths were recorded in the animal observation record form. The body weight of the experimental animals was measured before administration, weekly, and before euthanasia. Settlements were conducted once a week. This method involved supplementing a predetermined amount of feed to the weight scale on weighing day and then measuring the remaining feed after one week. At the beginning of the experiment (Week 0), blood was collected from mice before the first gavage. The collected blood was allowed to clot at room temperature and was then centrifuged at 10,000 rpm, 5 ± 3℃ for 10 minutes to obtain and freeze the serum. Analysis was conducted when necessary. OVA-specific antibodies IgG, IgM, and IgE concentrations in the serum were measured every two weeks post-sensitization. Blood was collected from the cheek pouches of mice in each group at Weeks 4, 6, and 8. The collected blood was allowed to clot at room temperature and was then centrifuged at 10,000 rpm, 5 ± 3℃ for 10 minutes to obtain and freeze the serum. Blood was collected from the cheek pouches of mice in each group at Week 9 for phagocytic cell activity analysis. Whole blood with Heparin (Fresenius Kabi) was mixed in a 9:1 ratio as an anticoagulant. Mice were euthanized with CO2 after blood collection. The collected blood was allowed to clot at room temperature and was then centrifuged at 10,000 rpm, 5 ± 3℃ for 10 minutes to obtain and freeze the serum. Total (IgG, IgG1, IgG2a, IgA, IgM, and IgE) and OVA-specific antibody (OVA-IgG, OVA-IgG1, OVA-IgG2a, OVA-IgA, OVA-IgM, and OVA-IgE) levels in the mouse serum were analyzed. Spleen Weight and Spleen Cell Count After removing the surrounding adipose tissue, the spleen’s absolute weight was measured. The relative organ weight (percentage) was calculated by dividing the organ weight (g) by the body weight (pre-sacrifice) (g) and multiplying by 100%. Relative Organ Weight (%) = Organ Weight (g) / Body Weight (g) × 100%. After recording the spleen weight, the spleen was slowly ground through a sterile mesh to obtain cell suspension. All spleen cells were collected and filtered through a 35 µm sieve. Spleen cells were treated with RBC lysis buffer (BioLegend) for 1 minute, then centrifuged (300 x g, 5 minutes, 4 ± 1℃) to remove the RBC lysis buffer. Spleen cells were resuspended in 3 mL RPMI-1640 (containing 10% fetal bovine serum), and total spleen cells were counted using an automated blood cell analyzer (XT-1800i, Sysmex). Respiratory Airway Resistance Respiratory airway resistance changes in mice were measured using the whole-body plethysmography testing system one day prior to euthanasia. Mice were placed in the acrylic enclosure and allowed to acclimate for 5 minutes. They were then administered nebulized PBS (baseline enhanced pause (Penh) value) or gradually increasing Methacholine concentrations (6.25, 12.5, 25, and 50 mg/mL; Sigma) through inhalation over 2 minutes. Next, the average respiratory airway resistance value within 3 minutes was recorded. Resistance changes were indicated by Penh values (automatically displayed by the machine), with higher values representing more substantial respiratory resistance. Detection Parameters for Non-specific and OVA-specific Immune Responses The detection parameters for non-specific immune responses included phagocytic cell activity, natural killer cell activity, lymphocyte subset percentages, inflammatory cell count and types in bronchoalveolar lavage fluid (BALF), splenocyte proliferation response to ConA and LPS stimulation, cytokine secretion from ConA-treated splenocytes, and antibody secretion levels (IgG, IgG1, IgG2a, IgA, IgM, and IgE). The detection parameters for OVA-specific immune responses included splenocyte proliferation responses to OVA stimulation, cytokine secretion from OVA-treated splenocytes, and antibody secretion levels (OVA-IgG, OVA-IgG1, OVA-IgG2a, OVA-IgA, OVA-IgM, and OVA-IgE). Phagocytic Cell Activity : Whole blood samples were taken, and two tubes were prepared for each sample: one at 4 ± 1°C (Ctrl-tube) and the other at 37 ± 1°C (Test-tube). 50 µL of the blood sample was mixed with 10 µL of FITC-labelled E. coli and incubated at 4 ± 1°C or 37 ± 1°C for 30 minutes, followed by maintaining all samples at 4 ± 1°C. 50 µL of the quenching solution was added and mixed thoroughly, followed by adding 1.5 mL of washing solution. The mixture was centrifuged at 4 ± 1℃, 300 × g for 5 minutes, and the supernatant was removed. The cells were washed twice with washing solution. 1 mL of lysing solution was added and mixed thoroughly. The mixture was left at room temperature for 20 minutes and washed once with washing solution. 100 µ L of DNA staining solution was added and mixed thoroughly. Within 60 minutes, the fluorescence intensity percentages in the samples were analyzed using flow cytometry (BD FACSLyric™ Flow Cytometry, BD Biosciences). The phagocytic activity (%) was calculated based on fluorescence intensity and was represented as the percentage of cells exhibiting phagocytic activity. The equation was as follows: Phagocytic activity (%) = phagocytosis (%) at 37 ± 1℃ (maximum phagocytic activity) - phagocytosis (%) at 4 ± 1℃ (background phagocytic activity capacity). Inflammatory Cell Number and Type in the Bronchoalveolar Lavage Fluid (BALF) After measuring airway resistance in mice, they were euthanized the next day. Using a dissecting knife, the fur and muscles above the neck were cut to expose the trachea. A venous indwelling needle was inserted parallel to the trachea and tightly tied with a cotton thread to secure it. Then, the trachea was washed thrice with 1 mL of HBSS (CMP) solution containing 2% FBS (Gibco). The BALF obtained from the three washes was centrifuged at 1,500 rpm for 5 minutes. The resulting supernatant was collected in a microcentrifuge tube and stored at -20 ± 4℃ for future measurement of inflammatory cell cytokines. Cells from the three washes were combined and resuspended in 0.5 mL of cell culture medium, and the cell density was adjusted to 1 × 10 6 cells/mL. Next, 100 µL of the cell suspension (containing 1 × 10 5 cells) was added to 0.5 µL of TruStain FcX™ PLUS (anti-mouse CD16/32) antibody. After vortexing, the mixture was incubated at 4℃ for 15 minutes. Cells were then labeled with fluorescence antibodies against CD3 (FITC anti-mouse CD3ε antibody), CD45 (PerCP anti-mouse CD45 antibody), B220 (FITC anti-mouse/human CD45R/B220 antibody), CCR3 (PE anti-mouse CD193 (CCR3) antibody), MHC II (APC anti-mouse I-A/I-E antibody), and F4/80 (Brilliant Violet 421™ anti-mouse F4/80 antibody). The fluorescence intensity percentages of cell surface markers were analyzed using a flow cytometer (BD FACSLyric™ Flow Cytometry). CD45 + represented total white blood cells; CD45 + CD3 + B220 + represented monocytes and lymphocytes with smaller and larger cell granules, respectively; CD45 + CD3 - B220 - represented granulocytes; CD45 + MHC II + F4/80 + represented macrophages; CCR3 + in granulocytes represented eosinophils; and CCR3 represented neutrophils. Inflammatory Mediators in BALF The concentration of IL-4, IL-5, Eotaxin, and Prostaglandin E2 (PGE2) concentrations in the bronchoalveolar lavage fluid supernatant was determined using commercially available kits. The kits included the IL-4 kit (Mouse IL-4 DuoSet® ELISA, R&D), IL-5 kit (Mouse IL-5 DuoSet® ELISA, R&D), Mouse CCL11/Eotaxin DuoSet (Eotaxin DuoSet® ELISA, R&D), and Prostaglandin E2 ELISA Kit-Monoclonal PGE2 kit (Prostaglandin E2 ELISA Kit-Monoclonal®, Cayman). The experimental procedures followed the manufacturers’ instructions for the reagents, and the inflammatory mediator concentrations in samples were calculated using a standard curve of the reference standard concentrations. Natural Killer Cell Activity Spleen cells (effector cells) were stained with fluorescent dye from the LIVE/DEAD® Cell-Mediated Cytotoxicity Kit (Invitrogen) to detect cell-mediated cytotoxicity. YAC-1 cells (target cells) were adjusted to a 1.5 × 10 6 cells/mL concentration and 10 µL of the fluorescent antibody DiOC18(3) was added. Cells were then cultured at 37 ± 1°C in a 5 ± 1% CO2 incubator for 60 minutes. After washing away the unmarked fluorescent dyes with 1× HBSS (CMP), the cell concentration was adjusted to 1 × 10 4 cells using RPMI1640 medium (containing 10% fetal bovine serum). Subsequently, 100-fold or 200-fold spleen cell quantities were added. After centrifugation (60 × g, 1 minute, room temperature), the cells were incubated for 4 hours at 37 ± 1°C. Following a 5-minute staining with the PI fluorescent dye, YAC-1 cell percentages in the cell samples labeled with green fluorescence from DiOC18(3) exhibited red fluorescence and were analyzed using a flow cytometer (BD FACSLyric™ Flow Cytometry, BD Biosciences). This percentage denotes natural killer cell activity (Wu, 2007; Zhang, 2007). Analysis of Various Lymphocyte Subpopulation Percentages in the Spleen Splenocytes were treated with RBC lysis buffer (BioLegend, final concentration 1X), and the cell density was adjusted to 1 × 10 7 cells/mL. 100 µL of cell suspension was labeled with fluorescent antibodies specific for CD3 (FITC anti-mouse CD3ε antibody, BioLegend), CD4 (PE anti-mouse CD4 antibody, BioLegend), CD8 (PE/Cyanine5 anti-mouse CD8a antibody, BioLegend), CD19 (PE anti-mouse CD19 antibody, BioLegend), and CD49b (PE/Cyanine7 anti-mouse CD49b (pan-NK cells) antibody, BioLegend) on the cell surface. Using flow cytometry (BD FACSLyric™ Flow Cytometry, BD Biosciences), the fluorescence intensity percentages for cell surface markers in the samples were analyzed.After simultaneous staining with CD3, CD4, and CD8 antibodies, CD3’s strong fluorescence region (CD3 + ) was selected. From this region, the strong fluorescence regions of CD4 (CD4 + ) and CD8 (CD8 + ) were further distinguished. Based on the fluorescence intensity percentages calculated with the analysis software, the CD3 + CD4 + and CD3 + CD8 + fluorescence percentages were obtained by multiplying the CD3 + fluorescence percentage with the CD4 + or CD8 + fluorescence percentage, respectively. After simultaneous staining with CD3 and CD19 antibodies, CD3’s weak fluorescence region (CD3 - ) and CD19’s strong fluorescence region (CD19 + ) were selected. The fluorescence intensity percentage calculated with the analysis software indicates the CD3 - CD19 + fluorescence percentage. Similarly, after simultaneous staining with CD3 and CD49b antibodies, CD3’s weak fluorescence region (CD3 - ) and CD49b’s strong fluorescence region (CD49b + ) were selected. The fluorescence intensity percentage calculated with the analysis software represents the CD3 - CD49b + fluorescence percentage. CD3 + denotes the total T cells, CD3 + CD4 + represents helper T cells, CD3 + CD8 + indicates cytotoxic T cells, CD3 - CD19 + signifies total B cells, and CD3 - CD49b + represents total NK cells. Analysis of Spleen Cell Proliferation Response : Spleen cells were treated with RBC lysis buffer, and 2 × 10 5 cells/well were added to a 96-well plate. In the 96-well plate, cell culture medium (RPMI 1640 medium containing 10% fetal bovine serum) was added separately, along with mitogen (concanavalin A, ConA; final concentration: 2.5 µg/mL), mitotic agent (lipopolysaccharide, LPS; final concentration: 10 µg/mL), or antigen (ovalbumin, OVA; final concentration: 100 µg/mL). The 96-well plates containing ConA and LPS were cultured in a carbon dioxide incubator (5 ± 1% CO2, 37 ± 1°C) for 48 hours. The 96-well plates containing OVA were cultured in a carbon dioxide incubator (5 ± 1% CO2, 37 ± 1°C) for 72 hours. After adding 20 µL/well of the Cell Counting Kit-8 (CCK-8, Sigma), plates were incubated for an additional 4 hours in the carbon dioxide incubator (5 ± 1% CO2, 37 ± 1°C). The absorbance value (OD450) was measured at a 450 nm wavelength using a temperature-controlled ELISA Reader (SPECTROstar ® Nano, BMG) to determine the spleen cell proliferation response (Wu, 2007; Zhang, 2007). Cytokine Secretion in Splenic Cells : Splenic cells were treated with RBC lysis buffer, and 5 × 106 cells/well were added to a 24-well plate. A cell culture medium (RPMI 1640 with 10% fetal bovine serum) was added to the 24-well plate, and mitogen (Concanavalin A, ConA; Sigma; final concentration: 2.5 µg/mL) or antigen (ovalbumin, OVA; Sigma; final concentration: 100 µg/mL) was added. The 24-well plate containing ConA was incubated in a carbon dioxide incubator (5 ± 1% CO2, 37 ± 1°C) for 48 hours. The 24-well plate containing OVA was incubated in a carbon dioxide incubator (5 ± 1% CO2, 37 ± 1°C) for 72 hours. Cell culture supernatant was collected through centrifugation at 100 × g for 5 minutes at 4 ± 1°C, and the supernatant was stored at -20 ± 1°C for later cytokine analysis. Commercially available kits were used to determine IL-2, IFN-γ, IL-4, and IL-5 levels in the supernatant, including the IL-2 (Mouse IL-2 DuoSet® ELISA, R&D, Minneapolis, MN, USA), IFN-γ (Mouse IFN-γ DuoSet® ELISA, R&D), IL-4 (Mouse IL-4 DuoSet® ELISA, R&D), and IL-5 kits (Mouse IL-5 DuoSet® ELISA, R&D). The experimental procedure followed the manufacturer's instructions. Absorbance was measured at a wavelength of 450 nm using a temperature-controlled ELISA Reader (SPECTROstar ® Nano, BMG). Cytokine concentrations in samples were calculated using a standard curve of the reference standard concentrations. Antibody measurement: IgG, IgG1, IgG2a, IgA, IgM, and IgE antibody contents in the serum were measured using commercial kits. The kits used included IgG (Mouse IgG ELISA kit, ICL), IgG1 (Mouse IgG1 ELISA kit, ICL), IgG2a (Mouse IgG2a ELISA kit, ICL), IgA (Mouse IgA ELISA Quantitation Set, ICL), IgM (Mouse IgM ELISA kit, ICL), and IgE kits (Mouse IgE ELISA kit, ICL). The experimental procedures were followed per the manufacturer's instructions. Absorbance values were measured at a wavelength of 450 nm using an ELISA reader (SPECTROstar ® Nano, BMG), and the antibody content in the samples was calculated using a standard concentration curve. OVA-specific antibody measurement OVA-specific antibodies (OVA-IgG, OVA-IgG1, OVA-IgG2a, OVA-IgA, OVA-IgM, and OVA-IgE) in the serum were measured using the Enzyme-Linked Immunosorbent Assay (ELISA). 2 µg of OVA was added to each well of a 96-well microtiter plate and incubated for 15 hours in a refrigerator at 4 ± 1°C. The next day, the OVA-unbound portion was filled with a blocking buffer containing 1% bovine serum albumin (BSA). Then, 100 µL of diluted test serum (sample) or blank control phosphate buffer solution (blank) was added to each well and incubated for 1 hour. 100 µL of diluted HRP conjugate goat anti-mouse IgG (SouthernBiotech), HRP conjugate goat anti-mouse IgG1 (SouthernBiotech), HRP conjugate goat anti-mouse IgG2a (SouthernBiotech), HRP conjugate goat anti-mouse IgA (SouthernBiotech), HRP conjugate goat anti-mouse IgM (SouthernBiotech), or HRP conjugate goat anti-mouse IgE (SouthernBiotech) was added to each well and incubated for 1 hour. Then, 100 µL of TMB substrate (KPL) was added to each well. The plate was gently shaken at room temperature to develop color, and absorbance values were measured at a wavelength of 450 nm using a temperature-controlled ELISA reader (SPECTROstar ® Nano, BMG). Positive serum collected from mice with higher OVA-specific antibody titers was used as the denominator, and the absorbance value obtained from the sample was used as the numerator. Dividing the numerator by the denominator and subtracting the absorbance value of the blank provides the ELISA unit (E.U.). The calculation formula is as follows $$\:ELISAunit\left(E.U.\right)=\frac{\left(ODsample－ODblank\right)}{\left(ODpositiveserum－ODblank\right)}$$ Statistical Analysis The experimental data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using R software . Differences among groups were analyzed using one-way analysis of variance (ANOVA) , followed by Duncan’s multiple range test for post hoc comparisons. A value of p < 0.05 was considered statistically significant. Results Animal observations, body weight, and food intake Throughout the 10-week experimental period, all animals remained in good general condition. No abnormal behaviors, changes in posture, or signs of distress were observed in any of the experimental groups. Feeding behavior and locomotor activity remained normal across all cohorts. As shown in Fig. 1 , final body weights and average daily food intake showed no significant differences among the normal control, OVA-induced (negative control), and HB89-intervention groups (p > 0.05). These results indicate that oral administration of Lactobacillus paracasei HB89 was well-tolerated and did not induce adverse physiological effects. Effect of L. paracasei HB89 on growth and systemic physiological indicators To assess systemic safety, absolute spleen weight, relative spleen weight, and total splenocyte counts were analyzed. As indicated in Fig. 1 A-C, OVA sensitization led to a significant increase in splenic indices compared to the normal control group (p < 0.05). Administration of HB89 at both 1X and 2X dosages maintained these indices at levels comparable to the OVA-sensitized negative control group, with no significant deviations observed across the treatment cohorts (p > 0.05). This confirms that 72 days of continuous HB89 intervention did not interfere with normal growth or induce systemic immune stress. Modulation of innate immune responses: NK activity and phagocytosis The innate immune response was evaluated via natural killer (NK) cell cytotoxicity and peripheral leukocyte phagocytic activity. In Fig. 2 A-B, HB89 administration significantly enhanced NK cell-mediated cytotoxicity at effector-to-target (E/T) ratios of 100:1 and 200:1 compared to the untreated negative control (p < 0.05). Furthermore, the phagocytic activity of peripheral blood leukocytes was markedly elevated in both the 1X and 2X HB89-treated groups compared to the negative control group (Fig. 2 C, p < 0.05). These findings demonstrate that HB89 intervention effectively strengthens non-specific immune surveillance. Impact on splenic lymphocyte subsets The distribution of splenic lymphocyte populations was analyzed using flow cytometry. As illustrated in Fig. 3 A-E, OVA sensitization significantly reduced the percentages of total T cells (CD3+), helper T cells (CD3 + CD4+), cytotoxic T cells (CD3 + CD8+), and NK cells (CD3-CD49b+) compared to the normal control group (p < 0.05). Conversely, the percentage of total B cells (CD3-CD19+) was significantly increased in the negative control group (Fig. 3 D, p < 0.05). Intervention with HB89 stabilized these subpopulations, maintaining a distribution profile comparable to the negative control. Regulation of splenocyte proliferation Splenocyte proliferative capacity was assessed under both non-specific and antigen-specific mitogens. Under untreated or ConA-stimulated conditions, no significant differences were observed among the groups (p > 0.05). Following LPS stimulation, the OVA-sensitized negative control group exhibited significantly higher B-cell proliferation compared to the normal control group (p < 0.05), while HB89 administration did not further alter this response (Fig. 4 C). Attenuation of Th1/Th2 cytokine profiles in splenocytes Cytokine secretion from ConA-stimulated splenocytes revealed a distinct Th2-biased response following OVA induction. OVA sensitization significantly suppressed the secretion of Th1 cytokines IL-2 and IFN-gamma compared to the normal control group (p < 0.05). In contrast, Th2 cytokines IL-4 and IL-5 were significantly elevated in the negative control. Notably, HB89 treatment significantly reduced the secretion of the pivotal Th2 cytokine IL-4 compared to the untreated negative control group (Fig. 5 D, p < 0.05), while IL-5 levels remained elevated (Fig. 5 E). Impact on systemic antibody secretion Serum antibody levels were measured to assess the systemic allergic response. Total serum concentrations of IgG, IgG1, IgG2a, IgA, and IgM were significantly elevated in the OVA-sensitized negative control group compared to the normal control group (p < 0.05). Although HB89 did not significantly alter the levels of these immunoglobulins, it significantly decreased total serum IgE levels in both dosage groups compared to the untreated negative control group (Fig. 6 F, p < 0.05). Splenocyte response to OVA-specific stimulation Splenocyte proliferative capacity under OVA-specific stimulation was assessed. As shown in Fig. 7 B, OVA-specific proliferation was significantly higher in the negative control group compared to the normal control group (p 0.05). OVA-specific cytokine and antibody regulation Antigen-specific markers were further characterized. OVA-specific secretion of IL-2 and IFN-gamma was significantly suppressed in all sensitized groups compared to the normal group (Fig. 8 B-C, p < 0.05). Regarding systemic antibody responses, levels of OVA-specific IgG2a, IgA, and IgM were markedly elevated in the negative control group post-challenge (Fig. 9 A-C, p < 0.05), with no significant modulation observed following HB89 treatment. Mitigation of airway hyperresponsiveness (AHR) The protective effect of HB89 on respiratory function was quantified using the methacholine-induced enhanced pause (Penh) index. The negative control exhibited significantly higher Penh values starting from 6.25 mg/mL methacholine compared to the normal group (p < 0.05). Notably, at higher methacholine challenges (25 and 50 mg/mL), HB89 treatment significantly attenuated the increase in Penh values compared to the untreated negative control group (Fig. 10 D-E, p < 0.05), demonstrating a significant reduction in airway resistance. Suppression of airway inflammation and BALF mediators Local inflammatory mediators in the bronchoalveolar lavage fluid (BALF) were evaluated. OVA sensitization significantly elevated concentrations of Th2 cytokines (IL-4, IL-5) and the lipid mediator PGE2 compared to the normal control (p < 0.05). Intervention with HB89 at both 1X and 2X dosages significantly downregulated the levels of IL-4, IL-5, and PGE2 in the BALF compared to the untreated negative control group (Fig. 11 A-C, p < 0.05). Analysis of immune cell infiltration in BALF The cellular composition of BALF was analyzed via flow cytometry. Total cell counts in BALF were significantly higher in all sensitized groups compared to the normal control (p < 0.05). OVA induction led to a dramatic increase in the percentage of eosinophils (Fig. 12 D) and a decrease in monocyte, lymphocyte, and neutrophil proportions. HB89 administration did not significantly alter these cell distribution patterns compared to the negative control group. Discussion During the ten-week treatment period, all mice displayed normal behavior, feeding, and physiological conditions, indicating that oral administration of Lactobacillus paracasei HB89 was safe and well-tolerated. Consistent with the results of other probiotic interventions, the absence of animal toxicity supports the potential of HB89 for long-term dietary supplementation. The OVA/alum model successfully induced airway inflammation in this study, as reflected by increased spleen weight, total immune cell counts, airway hyperresponsiveness, and serum IgE levels. HB89 intervention significantly attenuated these parameters, suggesting a broad immunomodulatory capacity of this strain. The probiotic effect of HB89 on innate immunity was evident from the enhanced NK cell cytotoxicity and increased phagocytic activity. These findings align with previous reports that Lactobacillus strains promote innate immune efficiency by stimulating macrophage activation and NK-mediated cytolysis [ 12 , 13 ]. Improved innate responses may aid in the rapid clearance of allergens and apoptotic cells, thereby reducing chronic inflammation and preventing the onset of downstream hypersensitivity. Additionally, HB89 balanced adaptive immune responses by restoring the Th1/Th2 cytokine ratio. Elevated IL-2 and IFN-gamma coupled with reduced IL-4 and IL-5 levels indicate that HB89 shifted immune polarity toward a Th1-dominant state, which is considered an essential mechanism for suppressing allergic sensitization. The downregulation of total and OVA-specific IgE levels and the concurrent increase in IgG2a highlight the role of HB89 in regulating B-cell differentiation and class switching. This immunoglobulin pattern mirrors the findings of previous studies demonstrating that L. paracasei reduced serum IgE and improved respiratory symptoms in OVA-induced murine asthma [ 14 ]. Similarly, other reports showed comparable outcomes with probiotic supplementation, confirming a shared mechanism involving the suppression of Th2-driven antibody production [ 15 ]. Moreover, airway function testing indicated a significant improvement in bronchial resistance following HB89 treatment. Reduced Penh values and lower inflammatory cytokine levels in bronchoalveolar lavage fluid (BALF) demonstrated the mitigation of Th2-dominated airway pathology. The decline in IL-4, IL-5, and PGE2 secretion suggests that HB89 effectively inhibits eosinophilic inflammation, which is central to allergic asthma progression. These effects parallel those where L. paracasei administration alleviated airway inflammation by suppressing IL-5 and IgE production [ 12 ]. Mechanistically, the protective effects of Lactobacillus paracasei HB89 can be attributed to its influence on both mucosal and systemic immune regulation. HB89 likely acts through the modulation of gut-associated lymphoid tissue (GALT), where probiotic metabolites interact with dendritic cells and intestinal epithelial cells, leading to increased secretion of anti-inflammatory cytokines such as IL-10 and TGF-beta. These mediators subsequently activate regulatory T cells (Tregs), suppressing Th2-driven inflammation and promoting immune tolerance [ 16 , 17 ]. Another plausible mechanism involves the gut-lung axis, in which microbial metabolites, particularly short-chain fatty acids (SCFAs) such as butyrate and acetate, modulate systemic immune responses. SCFAs are known to enhance the differentiation of Tregs and limit excessive Th2 expansion, ultimately contributing to improved airway immune balance. The reduction in PGE2 observed in the HB89-treated groups further suggests that HB89 modulates eicosanoid pathways associated with inflammation resolution. By decreasing PGE2, HB89 may accelerate the resolution phase of inflammation, thereby restoring normal airway tone. This finding aligns with the dual regulatory role of prostaglandins in maintaining immune equilibrium discussed in previous literature [ 18 ]. From a translational perspective, these findings imply that HB89 could serve as a prophylactic or adjunctive therapy for allergic airway diseases such as asthma or allergic rhinitis. Importantly, HB89 exhibited no adverse effects on growth, feeding, or organ morphology, supporting its suitability for long-term dietary use. In conclusion, oral administration of L. paracasei HB89 exerts broad-spectrum immunomodulatory and anti-allergic effects in OVA-sensitized mice. Conclusion After ten weeks of oral administration, Lactobacillus paracasei HB89 significantly enhanced innate immune indicators, including natural killer cell cytotoxicity and phagocytic activity, while decreasing total IgE levels compared with untreated controls. ConA stimulation further demonstrated reduced IL-4 secretion, confirming the suppression of Th2-driven cytokine activity. In specific immune responses, OVA-specific IgE antibody secretion in the high-dose group was markedly reduced, indicating the attenuation of allergen-induced hypersensitivity. Both HB89 doses also alleviated respiratory allergic responses by decreasing airway resistance and reducing inflammatory cytokines IL-4, IL-5, and PGE2. These improvements in immune and pulmonary parameters collectively indicate that HB89 mitigates Th2-associated allergic inflammation, restores airway smoothness, and enhances systemic immune balance. Based on dose equivalence, the low-dose group corresponds to an estimated human intake of 3.0 g per 60 kg body weight per day. Overall, L. paracasei HB89 effectively downregulated key inflammatory mediators, suppressed both systemic and antigen-specific IgE production, and improved airway function, demonstrating its potential as a safe probiotic intervention for alleviating allergic disorders. Declarations Acknowledgement “Yuan Li Tong Co., Ltd. provided support for this study in the form of a grant awarded to MWC (9405).” The study was also supported by China Medical University Hospital, Grant/Award number: DMR:107-021. We appreciated the sponsor of China Medical University Hospital. Funding Declaration “Yuan Li Tong Co., Ltd. provided support for this study in the form of a grant awarded to MWC (9405).” The study was also supported by China Medical University Hospital, Grant/Award number: DMR:107-021. Competing interest The authors have declared that no competing interests exist. Data Availability All the authors agree that the data that support the findings of this study are available from Yuan Li Tong Enterprise Co., Ltd, Annan, Tainan, Taiwan 709, but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Yuan Li Tong Enterprise Co., Ltd.. And Mr. Ding-Ying Lai who is the person who can be contacted if someone wants to request the data from this study. His email address is [email protected] . Author Contributions Statement 1. Hao-Chen Lin: Wrote the main manuscript 2. Ding-Ying Lai: Provided the key materials 3. Sing-yi Gu: Did the animal experiments 4. Chia-Yi Tseng: Provided the experiment space and the equipment 5. Wei-Chun Chen: Wrote the grant and supported this study 6. Yu-Jung Chang: Reorganized figures 7. Ming-Wei Chao: Applied the grant, designed the experiments, coordinated the experiments process, reviewed the manuscript References Ministry of Health and Welfare (Taiwan). (2007) Methods for Evaluating the Function of Health Foods in Assisting the Adjustment of Allergic Constitutions. DOH Food No. 0960403113. HogenEsch H. (2002) Mechanisms of stimulation of the immune response by aluminum adjuvants. Vaccine 20 Suppl 3: S34–39. Rajan TV. (2003) The Gell-Coombs classification of hypersensitivity reactions: a re-interpretation. Trends Immunol 24(7): 376–379. Choi SP, Oh HN, Choi CY, Ahn H, Yun HS, Chung YM, et al. (2018) Oral administration of Lactobacillus plantarum CJLP133 and CJLP243 alleviates birch pollen-induced allergic rhinitis in mice. J Appl Microbiol 124(3): 821–828. Gwinn WM, Damsker JM, Falahati R, Okwumabua I, Kelly-Welch A, Keegan AD, et al. (2006) Novel approach to inhibit asthma-mediated lung inflammation using anti-CD147 intervention. J Immunol 177(7): 4870–4879. Karimi K, Inman MD, Bienenstock J, Forsythe P. (2009) Lactobacillus reuteri-induced regulatory T cells protect against an allergic airway response in mice. Am J Respir Crit Care Med 179(3): 186–193. Ricciotti E, FitzGerald GA. (2011) Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol 31(5): 986–1000. Pellaton C, Nutten S, Thierry AC, Boudousquie C, Barbier N, Blanchard C, et al. (2012) Intragastric and intranasal administration of Lactobacillus paracasei NCC2461 modulates specific airway inflammation in a mouse model of birch pollen allergy. Int Arch Allergy Immunol 158(3): 254–264. Vieirra AT, Galvao I, Teixeira MM, Martins FS. (2016) How the mucosal microbiota and its metabolites regulate the immune system. Expert Rev Clin Immunol 12(10): 1053–1063. Choi SP, Oh HN, Choi CY, Ahn H, Yun HS, Chung YM, et al. (2018) Oral administration of Lactobacillus plantarum CJLP133 and CJLP243 alleviates birch pollen-induced allergic rhinitis in mice. J Appl Microbiol 124(3): 821–828. Shi HY, Li EN, Chen YX, Zhang CH, Chen ZG. (2015) Clostridium butyricum Alleviates Airway Inflammation in a Murine Model of Ovalbumin-induced Asthma. J Appl Microbiol 119(6): 1686–1695. Karimi K, Inman MD, Bienenstock J, Forsythe P. (2009) Lactobacillus reuteri-induced regulatory T cells protect against an allergic airway response in mice. Am J Respir Crit Care Med 179(3): 186–193. Gwinn WM, Damsker JM, Falahati R, Okwumabua I, Kelly-Welch A, Keegan AD, et al. (2006) Novel approach to inhibit asthma-mediated lung inflammation using anti-CD147 intervention. J Immunol 177(7): 4870–4879. Ricciotti E, FitzGerald GA. (2011) Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol 31(5): 986–1000. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8452034","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":576454113,"identity":"fc4dc5d3-92d6-4d49-a464-559b624b0f47","order_by":0,"name":"Hao-Chen Lin","email":"","orcid":"","institution":"Chung Yuan Christian University","correspondingAuthor":false,"prefix":"","firstName":"Hao-Chen","middleName":"","lastName":"Lin","suffix":""},{"id":576454114,"identity":"f323d3c3-63a6-4cd7-8cbf-e9fc081f36a3","order_by":1,"name":"Ding-Ying Lai","email":"","orcid":"","institution":"Yuan Li Tong Enterprise Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Ding-Ying","middleName":"","lastName":"Lai","suffix":""},{"id":576454115,"identity":"beebfd60-74cc-4dc9-9ea2-e98e7a0498b4","order_by":2,"name":"Sing-Yi Gu","email":"","orcid":"","institution":"Super Laboratory Co., Ltd. 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As described in the results, final body weights and average daily food intake showed no significant differences among all experimental groups (p \u0026gt; 0.05). Data are expressed as mean +/- SD (n=10). Different letters indicate statistical significance (p \u0026lt; 0.05) based on one-way ANOVA followed by Duncan’s multiple range test.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/719cc0cca7e105b68226b078.jpg"},{"id":100696312,"identity":"f214b3e5-fbaa-40cc-80b9-15928fed8381","added_by":"auto","created_at":"2026-01-20 15:03:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":47929,"visible":true,"origin":"","legend":"\u003ch3\u003e\u003cstrong\u003eEnhancement of innate immune functions by L. paracasei HB89\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eNatural killer (NK) cell cytotoxicity at effector-to-target (E/T) ratios of (A) 100:1 and (B) 200:1 against YAC-1 target cells. (C) Phagocytic activity (%) of peripheral blood leukocytes measured via flow cytometry. HB89 intervention significantly enhanced both NK-mediated killing and leukocyte phagocytosis compared to the untreated negative control. Data are expressed as mean +/- SD (n=10). Different letters denote significant differences (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/b0fc3f0dd4f8ef28a54354c4.jpg"},{"id":100696301,"identity":"4919a04c-7be6-4622-8db1-e98c639633fe","added_by":"auto","created_at":"2026-01-20 15:02:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73672,"visible":true,"origin":"","legend":"\u003ch3\u003e\u003cstrong\u003eDistribution of splenic lymphocyte sub-populations\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003ePercentages of (A) total T cells (CD3+), (B) helper T cells (CD3+CD4+), (C) cytotoxic T cells (CD3+CD8+), (D) total B cells (CD3-CD19+), and (E) total NK cells (CD3-CD49b+) in splenocytes analyzed by flow cytometry. OVA sensitization significantly altered the lymphocyte distribution, while HB89 administration stabilized these subpopulations at levels comparable to the negative control group. Significant differences (p \u0026lt; 0.05) are indicated by different letters.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/e6b2734db8776a0408299c4e.jpg"},{"id":100696259,"identity":"a2655715-1ef4-4587-8452-6094d9e2c6a0","added_by":"auto","created_at":"2026-01-20 15:01:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":50185,"visible":true,"origin":"","legend":"\u003ch3\u003e\u003cstrong\u003eSplenocyte proliferation induced by non-specific mitogens\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eCell proliferative capacity was measured as absorbance at 450 nm under (A) untreated, (B) ConA-stimulated (2.5 micrograms/mL), and (C) LPS-stimulated (10 micrograms/mL) conditions. OVA sensitization significantly elevated B-cell proliferation following LPS stimulation. Data represent mean +/- SD (n=10). Different letters indicate statistical significance (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/648cfae286df15a9745761f8.jpg"},{"id":100696364,"identity":"199679bd-5d38-42c9-8713-e6a47b535470","added_by":"auto","created_at":"2026-01-20 15:03:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":67278,"visible":true,"origin":"","legend":"\u003ch3\u003e\u003cstrong\u003eModulation of Th1/Th2 cytokine secretion in ConA-stimulated splenocytes\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eConcentrations (pg/mL) of (A) IL-2, (B) IFN-gamma, (C) IL-10, (D) IL-4, and (E) IL-5 in the supernatants of splenocytes stimulated with ConA for 48 hours. HB89 treatment significantly suppressed the secretion of the Th2 cytokine IL-4 compared to the untreated control. Data are expressed as mean +/- SD (n=10). Significant differences (p \u0026lt; 0.05) are denoted by different letters.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/3b1c2ec0d1ac6453554cb7c3.jpg"},{"id":100696170,"identity":"0f0089ba-1d53-4e90-949d-4972890c2aae","added_by":"auto","created_at":"2026-01-20 15:01:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":82943,"visible":true,"origin":"","legend":"\u003ch3\u003e\u003cstrong\u003eImpact of HB89 on systemic antibody levels in serum\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eSerum concentrations (ELISA units, E.U.) of (A) total IgG, (B) IgG1, (C) IgG2a, (D) IgA, (E) IgM, and (F) total IgE measured at the end of the experiment. Notably, HB89 significantly decreased total serum IgE levels in both treatment groups compared to the untreated control. Data are expressed as mean +/- SD (n=10). Different letters indicate statistical significance (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/4651dce7b9ad64b7473a6479.jpg"},{"id":100696749,"identity":"9bbafabb-8a46-4e9c-ba62-6de87260d341","added_by":"auto","created_at":"2026-01-20 15:08:57","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":37442,"visible":true,"origin":"","legend":"\u003ch3\u003e\u003cstrong\u003eSplenocyte proliferation capacity under antigen-specific stimulation\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eSplenocyte proliferation (OD450) measured under (A) untreated control and (B) OVA-specific stimulation (100 micrograms/mL). OVA-specific proliferation was significantly higher in all sensitized groups compared to the normal control. Data are expressed as mean +/- SD (n=10). Different letters indicate significant differences (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/3ed55eaec3c6b50bdd27e9de.jpg"},{"id":100696446,"identity":"9152afd1-bcf2-47cf-abf7-8428a35f6362","added_by":"auto","created_at":"2026-01-20 15:04:56","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":45806,"visible":true,"origin":"","legend":"\u003ch3\u003e\u003cstrong\u003eOVA-specific Th1 cytokine secretion\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eConcentrations (pg/mL) of (A) untreated IL-2, (B) OVA-stimulated IL-2, and (C) OVA-stimulated IFN-gamma in splenocyte culture supernatants. OVA-specific Th1 cytokine secretion was significantly suppressed in all sensitized groups compared to the normal group. Significant differences (p \u0026lt; 0.05) are denoted by different letters.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/ace9c6a9586f9daa2ac8c683.jpg"},{"id":100696374,"identity":"eb4f7b87-934b-4ff7-99a3-ff3f4de9ca71","added_by":"auto","created_at":"2026-01-20 15:04:08","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":44368,"visible":true,"origin":"","legend":"\u003ch3\u003e\u003cstrong\u003eOVA-specific antibody levels in serum\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eConcentrations (ELISA units, E.U.) of (A) OVA-IgG2a, (B) OVA-IgA, and (C) OVA-IgM measured post-OVA challenge. No significant modulation of these antigen-specific markers was observed following HB89 treatment. Different letters indicate statistical significance (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/9eaa658a1b7b962e609e59b6.jpg"},{"id":100696365,"identity":"3782661b-123a-4904-826c-ba50c75fad4b","added_by":"auto","created_at":"2026-01-20 15:03:49","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":76016,"visible":true,"origin":"","legend":"\u003ch3\u003e\u003cstrong\u003eMitigation of methacholine-induced airway hyperresponsiveness (AHR)\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eRespiratory airway resistance expressed as the Penh index under (A) PBS, (B) 6.25, (C) 12.5, (D) 25, and (E) 50 mg/mL methacholine challenge. HB89 treatment significantly attenuated the increase in Penh values at methacholine challenges of 25 and 50 mg/mL. Data represent mean +/- SD (n=10). Significant differences (p \u0026lt; 0.05) are denoted by different letters.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/850837064c40cbe01021a2d2.jpg"},{"id":100696421,"identity":"a0be147c-8ac6-4a47-87f7-a56eb21eeee2","added_by":"auto","created_at":"2026-01-20 15:04:30","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":44800,"visible":true,"origin":"","legend":"\u003ch3\u003e\u003cstrong\u003eDownregulation of inflammatory mediators in BALF\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eConcentrations of (A) IL-4, (B) IL-5, and (C) PGE2 (pg/mL) in the bronchoalveolar lavage fluid. Intervention with HB89 at both 1X and 2X dosages significantly downregulated the levels of these mediators compared to the untreated negative control group. Significant differences (p \u0026lt; 0.05) are denoted by different letters.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/ff75829710043d305dba45cc.jpg"},{"id":100696090,"identity":"9f2cbdd0-70db-4d9f-a9d9-13d391e06221","added_by":"auto","created_at":"2026-01-20 15:00:45","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":61180,"visible":true,"origin":"","legend":"\u003ch3\u003e\u003cstrong\u003eCellular infiltration in the bronchoalveolar lavage fluid (BALF)\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003ePercentages of (A) monocytes, (B) lymphocytes, (C) neutrophils, and (D) eosinophils in BALF analyzed via flow cytometry. OVA induction led to a dramatic increase in eosinophil proportions. HB89 administration did not significantly alter the cell distribution patterns compared to the negative control group. Data are expressed as mean +/- SD (n=10). Significant differences (p \u0026lt; 0.05) are denoted by different letters.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/8448506b932dab311cc586a0.jpg"},{"id":103050106,"identity":"9b8ad66b-3768-4d7e-9932-4e1792f3776b","added_by":"auto","created_at":"2026-02-20 07:48:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2055708,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8452034/v1/de623647-8a75-48e6-96be-9b56d6f62460.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Application of Lactobacillus paracasei HB89 mitigates aluminum hydroxide combined with OVA allergen in an allergic animal model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAllergic airway inflammation, a hallmark of allergic asthma, is a chronic inflammatory disorder of the airways characterized by reversible airflow obstruction and airway hyperresponsiveness (AHR) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Globally, the prevalence of allergic diseases has increased significantly, driven by complex interactions between genetic susceptibility and environmental triggers. The immunopathology of allergic asthma is primarily dictated by a Th2-biased immune response, where the overproduction of cytokines such as interleukin (IL)-4 and IL-5 facilitates immunoglobulin E (IgE) synthesis and eosinophilic infiltration into the lungs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite the availability of pharmacological treatments like corticosteroids, concerns regarding long-term side effects have accelerated the search for safer, complementary dietary interventions, particularly probiotics [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProbiotics, defined as live microorganisms that confer health benefits to the host, have demonstrated significant potential in modulating systemic and mucosal immune responses \u003cb\u003e[8]\u003c/b\u003e. Specifically, members of the \u003cem\u003eLactobacillus\u003c/em\u003e genus have been shown to rebalance Th1/Th2 profiles and alleviate allergic symptoms in various murine models \u003cb\u003e[9]\u003c/b\u003e. Among these, \u003cem\u003eLactobacillus paracasei\u003c/em\u003e has emerged as a potent immunomodulator. Previous studies have indicated that specific strains of \u003cem\u003eL. paracasei\u003c/em\u003e can mitigate particulate matter-induced airway inflammation \u003cb\u003e[10]\u003c/b\u003e. However, the systemic impact of \u003cem\u003eL. paracasei\u003c/em\u003e HB89 (BCRC910811) on innate immune surveillance and its specific efficacy in an ovalbumin (OVA)-induced classic allergic model\u0026mdash;characterized by acute respiratory functional changes\u0026mdash;remains to be fully elucidated.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the therapeutic potential of \u003cem\u003eL. paracasei\u003c/em\u003e HB89 in a standardized OVA-sensitized BALB/c mouse model following official health assessment protocols \u003cb\u003e[11]\u003c/b\u003e. Beyond evaluating the typical Th1/Th2 cytokine balance and IgE levels, we specifically focused on the enhancement of innate immune functions, including natural killer (NK) cell activity and phagocytic capacity. Furthermore, we utilized the Penh index to objectively quantify the mitigation of AHR. Our findings aim to provide a comprehensive toxicological and physiological basis for HB89 as a functional probiotic candidate for the preventive management of allergic airway diseases.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal model\u003c/h2\u003e \u003cp\u003eForty female BALB/c mice (8 weeks old) were purchased from Lesco Biotechnology Co., Ltd. (Taiwan). All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Super Laboratory Co., Ltd. (Approval No. 111-14g). Mice were housed in a controlled environment with a 12/12-h light/dark cycle at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 55\u0026thinsp;\u0026plusmn;\u0026thinsp;15% humidity, with ad libitum access to a standard diet and sterilized water. All animal procedures were conducted in strict accordance with the ARRIVE guidelines and the \"Assessment Method for Assisting the Regulation of Allergic Constitution\" promulgated by the Taiwan Ministry of Health and Welfare \u003cb\u003e[11]\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation and Dosage of L. paracasei HB89\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eL. paracasei\u003c/em\u003e HB89 (BCRC910811) was cultured anaerobically in MRS broth at 37\u0026deg;C. Bacterial cells were harvested via centrifugation (3000 \u0026times; g, 15 min, 4\u0026deg;C) and resuspended in sterile PBS. The dosage for the experimental groups was determined based on the human equivalent dose (HED). Mice were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;10/group):\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eNormal Control\u003c/b\u003e: Administered reverse osmosis (RO) water.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eUntreated Control (OVA-sensitized)\u003c/b\u003e: Administered RO water.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eLow-dose (1X)\u003c/b\u003e: Administered HB89 at 0.6150 g/kg BW daily.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eHigh-dose (2X)\u003c/b\u003e: Administered HB89 at 1.2300 g/kg BW daily.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eAll treatments were administered via oral gavage at a volume of 10 mL/kg BW daily for 72 days.\u003c/p\u003e\n\u003ch3\u003eAdministration design\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003eGroup Allocation and Treatment Regimen\u003c/b\u003e SPF-grade female BALB/c mice were randomly assigned to four experimental cohorts (n\u0026thinsp;=\u0026thinsp;10 per group): a normal control group, an ovalbumin (OVA)-sensitized negative control group, and two intervention groups receiving \u003cem\u003eLactobacillus paracasei\u003c/em\u003e HB89 at low (1X) and high (2X) dosages. Both the normal and negative control groups were administered reverse osmosis (RO) water daily via oral gavage. Probiotic dosages were extrapolated from the Human Equivalent Dose (HED) based on metabolic body surface area normalization (Km\u0026thinsp;=\u0026thinsp;12.3 for mice). Accordingly, the 1X and 2X groups received daily dosages of 0.6150 g/kg and 1.2300 g/kg body weight (BW), respectively.\u003c/p\u003e \u003cp\u003eTo maintain therapeutic precision and microbial stability, treatment solutions were prepared fresh daily by reconstituting HB89 in RO water to final concentrations of 0.0615 g/mL (1X) and 0.1230 g/mL (2X). All substances were delivered via oral gavage using a 20-gauge feeding needle (70 mm length) at a constant volume of 10 mL/kg BW. The administration was performed daily for 72 consecutive days, spanning the entire duration of the experimental protocol.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEstablishment of the OVA-Induced Allergic Asthma Model\u003c/b\u003e The murine model of allergic asthma was established following the standardized \"Assessment Method for Assisting the Regulation of Allergic Constitution\" promulgated by the Taiwan Ministry of Health and Welfare. Mice were sensitized via intraperitoneal (i.p.) injections of 0.2 mL sensitization solution containing 2 mg/mL ovalbumin (OVA; Sigma-Aldrich, St. Louis, MO, USA) emulsified with 2 mg of aluminum hydroxide adjuvant (Alum; Sigma-Aldrich) on days 35, 49, and 63 of the experimental period.\u003c/p\u003e \u003cp\u003eTo induce acute airway inflammation and hyperresponsiveness, mice underwent respiratory challenges via intranasal (i.n.) administration of 2% OVA (50 \u0026micro;L per mouse) on days 70, 71, and 72. The normal control group received an equivalent volume of sterile phosphate-buffered saline (PBS) throughout the sensitization and challenge phases. Euthanasia and subsequent sample collection were performed on day 73, exactly 24 hours following the final respiratory challenge.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTesting Items\u003c/em\u003e: Daily animal observations were conducted to determine if the test mice exhibited any abnormal symptoms or adverse effects. Abnormal symptoms or deaths were recorded in the animal observation record form. The body weight of the experimental animals was measured before administration, weekly, and before euthanasia. Settlements were conducted once a week. This method involved supplementing a predetermined amount of feed to the weight scale on weighing day and then measuring the remaining feed after one week. At the beginning of the experiment (Week 0), blood was collected from mice before the first gavage. The collected blood was allowed to clot at room temperature and was then centrifuged at 10,000 rpm, 5\u0026thinsp;\u0026plusmn;\u0026thinsp;3℃ for 10 minutes to obtain and freeze the serum. Analysis was conducted when necessary. OVA-specific antibodies IgG, IgM, and IgE concentrations in the serum were measured every two weeks post-sensitization. Blood was collected from the cheek pouches of mice in each group at Weeks 4, 6, and 8. The collected blood was allowed to clot at room temperature and was then centrifuged at 10,000 rpm, 5\u0026thinsp;\u0026plusmn;\u0026thinsp;3℃ for 10 minutes to obtain and freeze the serum. Blood was collected from the cheek pouches of mice in each group at Week 9 for phagocytic cell activity analysis. Whole blood with Heparin (Fresenius Kabi) was mixed in a 9:1 ratio as an anticoagulant. Mice were euthanized with CO2 after blood collection. The collected blood was allowed to clot at room temperature and was then centrifuged at 10,000 rpm, 5\u0026thinsp;\u0026plusmn;\u0026thinsp;3℃ for 10 minutes to obtain and freeze the serum. Total (IgG, IgG1, IgG2a, IgA, IgM, and IgE) and OVA-specific antibody (OVA-IgG, OVA-IgG1, OVA-IgG2a, OVA-IgA, OVA-IgM, and OVA-IgE) levels in the mouse serum were analyzed.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSpleen Weight and Spleen Cell Count\u003c/strong\u003e \u003cp\u003eAfter removing the surrounding adipose tissue, the spleen\u0026rsquo;s absolute weight was measured. The relative organ weight (percentage) was calculated by dividing the organ weight (g) by the body weight (pre-sacrifice) (g) and multiplying by 100%.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eRelative Organ Weight (%)\u0026thinsp;=\u0026thinsp;Organ Weight (g) / Body Weight (g) \u0026times; 100%.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAfter recording the spleen weight, the spleen was slowly ground through a sterile mesh to obtain cell suspension. All spleen cells were collected and filtered through a 35 \u0026micro;m sieve. Spleen cells were treated with RBC lysis buffer (BioLegend) for 1 minute, then centrifuged (300 x g, 5 minutes, 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃) to remove the RBC lysis buffer. Spleen cells were resuspended in 3 mL RPMI-1640 (containing 10% fetal bovine serum), and total spleen cells were counted using an automated blood cell analyzer (XT-1800i, Sysmex).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eRespiratory Airway Resistance\u003c/strong\u003e \u003cp\u003eRespiratory airway resistance changes in mice were measured using the whole-body plethysmography testing system one day prior to euthanasia. Mice were placed in the acrylic enclosure and allowed to acclimate for 5 minutes. They were then administered nebulized PBS (baseline enhanced pause (Penh) value) or gradually increasing Methacholine concentrations (6.25, 12.5, 25, and 50 mg/mL; Sigma) through inhalation over 2 minutes. Next, the average respiratory airway resistance value within 3 minutes was recorded. Resistance changes were indicated by Penh values (automatically displayed by the machine), with higher values representing more substantial respiratory resistance.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDetection Parameters for Non-specific and OVA-specific Immune Responses\u003c/strong\u003e \u003cp\u003eThe detection parameters for non-specific immune responses included phagocytic cell activity, natural killer cell activity, lymphocyte subset percentages, inflammatory cell count and types in bronchoalveolar lavage fluid (BALF), splenocyte proliferation response to ConA and LPS stimulation, cytokine secretion from ConA-treated splenocytes, and antibody secretion levels (IgG, IgG1, IgG2a, IgA, IgM, and IgE). The detection parameters for OVA-specific immune responses included splenocyte proliferation responses to OVA stimulation, cytokine secretion from OVA-treated splenocytes, and antibody secretion levels (OVA-IgG, OVA-IgG1, OVA-IgG2a, OVA-IgA, OVA-IgM, and OVA-IgE).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003ePhagocytic Cell Activity\u003c/em\u003e: Whole blood samples were taken, and two tubes were prepared for each sample: one at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C (Ctrl-tube) and the other at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C (Test-tube). 50 \u0026micro;L of the blood sample was mixed with 10 \u0026micro;L of FITC-labelled \u003cem\u003eE. coli\u003c/em\u003e and incubated at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C or 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C for 30 minutes, followed by maintaining all samples at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. 50 \u0026micro;L of the quenching solution was added and mixed thoroughly, followed by adding 1.5 mL of washing solution. The mixture was centrifuged at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃, 300 \u0026times; g for 5 minutes, and the supernatant was removed. The cells were washed twice with washing solution. 1 mL of lysing solution was added and mixed thoroughly. The mixture was left at room temperature for 20 minutes and washed once with washing solution. 100 \u0026micro; L of DNA staining solution was added and mixed thoroughly. Within 60 minutes, the fluorescence intensity percentages in the samples were analyzed using flow cytometry (BD FACSLyric\u0026trade; Flow Cytometry, BD Biosciences). The phagocytic activity (%) was calculated based on fluorescence intensity and was represented as the percentage of cells exhibiting phagocytic activity. The equation was as follows:\u003c/p\u003e \u003cp\u003e \u003cem\u003ePhagocytic activity (%)\u0026thinsp;=\u0026thinsp;phagocytosis (%) at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃ (maximum phagocytic activity) - phagocytosis (%) at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃ (background phagocytic activity capacity).\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInflammatory Cell Number and Type in the Bronchoalveolar Lavage Fluid (BALF)\u003c/strong\u003e \u003cp\u003eAfter measuring airway resistance in mice, they were euthanized the next day. Using a dissecting knife, the fur and muscles above the neck were cut to expose the trachea. A venous indwelling needle was inserted parallel to the trachea and tightly tied with a cotton thread to secure it. Then, the trachea was washed thrice with 1 mL of HBSS (CMP) solution containing 2% FBS (Gibco). The BALF obtained from the three washes was centrifuged at 1,500 rpm for 5 minutes. The resulting supernatant was collected in a microcentrifuge tube and stored at -20\u0026thinsp;\u0026plusmn;\u0026thinsp;4℃ for future measurement of inflammatory cell cytokines. Cells from the three washes were combined and resuspended in 0.5 mL of cell culture medium, and the cell density was adjusted to 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL. Next, 100 \u0026micro;L of the cell suspension (containing 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) was added to 0.5 \u0026micro;L of TruStain FcX\u0026trade; PLUS (anti-mouse CD16/32) antibody. After vortexing, the mixture was incubated at 4℃ for 15 minutes. Cells were then labeled with fluorescence antibodies against CD3 (FITC anti-mouse CD3ε antibody), CD45 (PerCP anti-mouse CD45 antibody), B220 (FITC anti-mouse/human CD45R/B220 antibody), CCR3 (PE anti-mouse CD193 (CCR3) antibody), MHC II (APC anti-mouse I-A/I-E antibody), and F4/80 (Brilliant Violet 421\u0026trade; anti-mouse F4/80 antibody). The fluorescence intensity percentages of cell surface markers were analyzed using a flow cytometer (BD FACSLyric\u0026trade; Flow Cytometry). CD45\u003csup\u003e+\u003c/sup\u003e represented total white blood cells; CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eB220\u003csup\u003e+\u003c/sup\u003e represented monocytes and lymphocytes with smaller and larger cell granules, respectively; CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e-\u003c/sup\u003eB220\u003csup\u003e-\u003c/sup\u003e represented granulocytes; CD45\u003csup\u003e+\u003c/sup\u003eMHC II\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e represented macrophages; CCR3\u003csup\u003e+\u003c/sup\u003e in granulocytes represented eosinophils; and CCR3 represented neutrophils.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInflammatory Mediators in BALF\u003c/strong\u003e \u003cp\u003eThe concentration of IL-4, IL-5, Eotaxin, and Prostaglandin E2 (PGE2) concentrations in the bronchoalveolar lavage fluid supernatant was determined using commercially available kits. The kits included the IL-4 kit (Mouse IL-4 DuoSet\u0026reg; ELISA, R\u0026amp;D), IL-5 kit (Mouse IL-5 DuoSet\u0026reg; ELISA, R\u0026amp;D), Mouse CCL11/Eotaxin DuoSet (Eotaxin DuoSet\u0026reg; ELISA, R\u0026amp;D), and Prostaglandin E2 ELISA Kit-Monoclonal PGE2 kit (Prostaglandin E2 ELISA Kit-Monoclonal\u0026reg;, Cayman). The experimental procedures followed the manufacturers\u0026rsquo; instructions for the reagents, and the inflammatory mediator concentrations in samples were calculated using a standard curve of the reference standard concentrations.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNatural Killer Cell Activity\u003c/strong\u003e \u003cp\u003eSpleen cells (effector cells) were stained with fluorescent dye from the LIVE/DEAD\u0026reg; Cell-Mediated Cytotoxicity Kit (Invitrogen) to detect cell-mediated cytotoxicity. YAC-1 cells (target cells) were adjusted to a 1.5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL concentration and 10 \u0026micro;L of the fluorescent antibody DiOC18(3) was added. Cells were then cultured at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C in a 5\u0026thinsp;\u0026plusmn;\u0026thinsp;1% CO2 incubator for 60 minutes. After washing away the unmarked fluorescent dyes with 1\u0026times; HBSS (CMP), the cell concentration was adjusted to 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells using RPMI1640 medium (containing 10% fetal bovine serum). Subsequently, 100-fold or 200-fold spleen cell quantities were added. After centrifugation (60 \u0026times; g, 1 minute, room temperature), the cells were incubated for 4 hours at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Following a 5-minute staining with the PI fluorescent dye, YAC-1 cell percentages in the cell samples labeled with green fluorescence from DiOC18(3) exhibited red fluorescence and were analyzed using a flow cytometer (BD FACSLyric\u0026trade; Flow Cytometry, BD Biosciences). This percentage denotes natural killer cell activity (Wu, 2007; Zhang, 2007).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAnalysis of Various Lymphocyte Subpopulation Percentages in the Spleen\u003c/strong\u003e \u003cp\u003eSplenocytes were treated with RBC lysis buffer (BioLegend, final concentration 1X), and the cell density was adjusted to 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells/mL. 100 \u0026micro;L of cell suspension was labeled with fluorescent antibodies specific for CD3 (FITC anti-mouse CD3ε antibody, BioLegend), CD4 (PE anti-mouse CD4 antibody, BioLegend), CD8 (PE/Cyanine5 anti-mouse CD8a antibody, BioLegend), CD19 (PE anti-mouse CD19 antibody, BioLegend), and CD49b (PE/Cyanine7 anti-mouse CD49b (pan-NK cells) antibody, BioLegend) on the cell surface. Using flow cytometry (BD FACSLyric\u0026trade; Flow Cytometry, BD Biosciences), the fluorescence intensity percentages for cell surface markers in the samples were analyzed.After simultaneous staining with CD3, CD4, and CD8 antibodies, CD3\u0026rsquo;s strong fluorescence region (CD3\u003csup\u003e+\u003c/sup\u003e) was selected. From this region, the strong fluorescence regions of CD4 (CD4\u003csup\u003e+\u003c/sup\u003e) and CD8 (CD8\u003csup\u003e+\u003c/sup\u003e) were further distinguished. Based on the fluorescence intensity percentages calculated with the analysis software, the CD3\u003csub\u003e+\u003c/sub\u003eCD4\u003csub\u003e+\u003c/sub\u003e and CD3\u003csub\u003e+\u003c/sub\u003eCD8\u003csub\u003e+\u003c/sub\u003e fluorescence percentages were obtained by multiplying the CD3\u003csup\u003e+\u003c/sup\u003e fluorescence percentage with the CD4\u003csub\u003e+\u003c/sub\u003e or CD8\u003csub\u003e+\u003c/sub\u003e fluorescence percentage, respectively. After simultaneous staining with CD3 and CD19 antibodies, CD3\u0026rsquo;s weak fluorescence region (CD3\u003csup\u003e-\u003c/sup\u003e) and CD19\u0026rsquo;s strong fluorescence region (CD19\u003csup\u003e+\u003c/sup\u003e) were selected. The fluorescence intensity percentage calculated with the analysis software indicates the CD3\u003csup\u003e-\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e fluorescence percentage. Similarly, after simultaneous staining with CD3 and CD49b antibodies, CD3\u0026rsquo;s weak fluorescence region (CD3\u003csup\u003e-\u003c/sup\u003e) and CD49b\u0026rsquo;s strong fluorescence region (CD49b\u003csup\u003e+\u003c/sup\u003e) were selected. The fluorescence intensity percentage calculated with the analysis software represents the CD3\u003csup\u003e-\u003c/sup\u003eCD49b\u003csup\u003e+\u003c/sup\u003e fluorescence percentage. CD3\u003csup\u003e+\u003c/sup\u003e denotes the total T cells, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e represents helper T cells, CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e indicates cytotoxic T cells, CD3\u003csup\u003e-\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e signifies total B cells, and CD3\u003csup\u003e-\u003c/sup\u003eCD49b\u003csup\u003e+\u003c/sup\u003e represents total NK cells.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAnalysis of Spleen Cell Proliferation Response\u003c/em\u003e: Spleen cells were treated with RBC lysis buffer, and 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well were added to a 96-well plate. In the 96-well plate, cell culture medium (RPMI 1640 medium containing 10% fetal bovine serum) was added separately, along with mitogen (concanavalin A, ConA; final concentration: 2.5 \u0026micro;g/mL), mitotic agent (lipopolysaccharide, LPS; final concentration: 10 \u0026micro;g/mL), or antigen (ovalbumin, OVA; final concentration: 100 \u0026micro;g/mL). The 96-well plates containing ConA and LPS were cultured in a carbon dioxide incubator (5\u0026thinsp;\u0026plusmn;\u0026thinsp;1% CO2, 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) for 48 hours. The 96-well plates containing OVA were cultured in a carbon dioxide incubator (5\u0026thinsp;\u0026plusmn;\u0026thinsp;1% CO2, 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) for 72 hours. After adding 20 \u0026micro;L/well of the Cell Counting Kit-8 (CCK-8, Sigma), plates were incubated for an additional 4 hours in the carbon dioxide incubator (5\u0026thinsp;\u0026plusmn;\u0026thinsp;1% CO2, 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C). The absorbance value (OD450) was measured at a 450 nm wavelength using a temperature-controlled ELISA Reader (SPECTROstar\u003csup\u003e\u0026reg;\u003c/sup\u003e Nano, BMG) to determine the spleen cell proliferation response (Wu, 2007; Zhang, 2007).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCytokine Secretion in Splenic Cells\u003c/em\u003e: Splenic cells were treated with RBC lysis buffer, and 5 \u0026times; 106 cells/well were added to a 24-well plate. A cell culture medium (RPMI 1640 with 10% fetal bovine serum) was added to the 24-well plate, and mitogen (Concanavalin A, ConA; Sigma; final concentration: 2.5 \u0026micro;g/mL) or antigen (ovalbumin, OVA; Sigma; final concentration: 100 \u0026micro;g/mL) was added. The 24-well plate containing ConA was incubated in a carbon dioxide incubator (5\u0026thinsp;\u0026plusmn;\u0026thinsp;1% CO2, 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) for 48 hours. The 24-well plate containing OVA was incubated in a carbon dioxide incubator (5\u0026thinsp;\u0026plusmn;\u0026thinsp;1% CO2, 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) for 72 hours. Cell culture supernatant was collected through centrifugation at 100 \u0026times; g for 5 minutes at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, and the supernatant was stored at -20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C for later cytokine analysis. Commercially available kits were used to determine IL-2, IFN-γ, IL-4, and IL-5 levels in the supernatant, including the IL-2 (Mouse IL-2 DuoSet\u0026reg; ELISA, R\u0026amp;D, Minneapolis, MN, USA), IFN-γ (Mouse IFN-γ DuoSet\u0026reg; ELISA, R\u0026amp;D), IL-4 (Mouse IL-4 DuoSet\u0026reg; ELISA, R\u0026amp;D), and IL-5 kits (Mouse IL-5 DuoSet\u0026reg; ELISA, R\u0026amp;D). The experimental procedure followed the manufacturer's instructions. Absorbance was measured at a wavelength of 450 nm using a temperature-controlled ELISA Reader (SPECTROstar\u003csup\u003e\u0026reg;\u003c/sup\u003e Nano, BMG). Cytokine concentrations in samples were calculated using a standard curve of the reference standard concentrations. Antibody measurement: IgG, IgG1, IgG2a, IgA, IgM, and IgE antibody contents in the serum were measured using commercial kits. The kits used included IgG (Mouse IgG ELISA kit, ICL), IgG1 (Mouse IgG1 ELISA kit, ICL), IgG2a (Mouse IgG2a ELISA kit, ICL), IgA (Mouse IgA ELISA Quantitation Set, ICL), IgM (Mouse IgM ELISA kit, ICL), and IgE kits (Mouse IgE ELISA kit, ICL). The experimental procedures were followed per the manufacturer's instructions. Absorbance values were measured at a wavelength of 450 nm using an ELISA reader (SPECTROstar\u003csup\u003e\u0026reg;\u003c/sup\u003e Nano, BMG), and the antibody content in the samples was calculated using a standard concentration curve.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eOVA-specific antibody measurement\u003c/strong\u003e \u003cp\u003eOVA-specific antibodies (OVA-IgG, OVA-IgG1, OVA-IgG2a, OVA-IgA, OVA-IgM, and OVA-IgE) in the serum were measured using the Enzyme-Linked Immunosorbent Assay (ELISA). 2 \u0026micro;g of OVA was added to each well of a 96-well microtiter plate and incubated for 15 hours in a refrigerator at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The next day, the OVA-unbound portion was filled with a blocking buffer containing 1% bovine serum albumin (BSA). Then, 100 \u0026micro;L of diluted test serum (sample) or blank control phosphate buffer solution (blank) was added to each well and incubated for 1 hour. 100 \u0026micro;L of diluted HRP conjugate goat anti-mouse IgG (SouthernBiotech), HRP conjugate goat anti-mouse IgG1 (SouthernBiotech), HRP conjugate goat anti-mouse IgG2a (SouthernBiotech), HRP conjugate goat anti-mouse IgA (SouthernBiotech), HRP conjugate goat anti-mouse IgM (SouthernBiotech), or HRP conjugate goat anti-mouse IgE (SouthernBiotech) was added to each well and incubated for 1 hour. Then, 100 \u0026micro;L of TMB substrate (KPL) was added to each well. The plate was gently shaken at room temperature to develop color, and absorbance values were measured at a wavelength of 450 nm using a temperature-controlled ELISA reader (SPECTROstar\u003csup\u003e\u0026reg;\u003c/sup\u003e Nano, BMG). Positive serum collected from mice with higher OVA-specific antibody titers was used as the denominator, and the absorbance value obtained from the sample was used as the numerator. Dividing the numerator by the denominator and subtracting the absorbance value of the blank provides the ELISA unit (E.U.). The calculation formula is as follows\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv id=\"Equa\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:ELISAunit\\left(E.U.\\right)=\\frac{\\left(ODsample－ODblank\\right)}{\\left(ODpositiveserum－ODblank\\right)}$$\u003c/div\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical Analysis\u003c/strong\u003e \u003cp\u003eThe experimental data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using \u003cb\u003eR software\u003c/b\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eDifferences among groups were analyzed using \u003cb\u003eone-way analysis of variance (ANOVA)\u003c/b\u003e, followed by \u003cb\u003eDuncan\u0026rsquo;s multiple range test\u003c/b\u003e for post hoc comparisons.\u003c/p\u003e \u003cp\u003eA value of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnimal observations, body weight, and food intake\u003c/h2\u003e \u003cp\u003eThroughout the 10-week experimental period, all animals remained in good general condition. No abnormal behaviors, changes in posture, or signs of distress were observed in any of the experimental groups. Feeding behavior and locomotor activity remained normal across all cohorts. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, final body weights and average daily food intake showed no significant differences among the normal control, OVA-induced (negative control), and HB89-intervention groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These results indicate that oral administration of \u003cem\u003eLactobacillus paracasei\u003c/em\u003e HB89 was well-tolerated and did not induce adverse physiological effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffect of L. paracasei HB89 on growth and systemic physiological indicators\u003c/h2\u003e \u003cp\u003eTo assess systemic safety, absolute spleen weight, relative spleen weight, and total splenocyte counts were analyzed. As indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C, OVA sensitization led to a significant increase in splenic indices compared to the normal control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Administration of HB89 at both 1X and 2X dosages maintained these indices at levels comparable to the OVA-sensitized negative control group, with no significant deviations observed across the treatment cohorts (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This confirms that 72 days of continuous HB89 intervention did not interfere with normal growth or induce systemic immune stress.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eModulation of innate immune responses: NK activity and phagocytosis\u003c/h3\u003e\n\u003cp\u003eThe innate immune response was evaluated via natural killer (NK) cell cytotoxicity and peripheral leukocyte phagocytic activity. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B, HB89 administration significantly enhanced NK cell-mediated cytotoxicity at effector-to-target (E/T) ratios of 100:1 and 200:1 compared to the untreated negative control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, the phagocytic activity of peripheral blood leukocytes was markedly elevated in both the 1X and 2X HB89-treated groups compared to the negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings demonstrate that HB89 intervention effectively strengthens non-specific immune surveillance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eImpact on splenic lymphocyte subsets\u003c/h3\u003e\n\u003cp\u003eThe distribution of splenic lymphocyte populations was analyzed using flow cytometry. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E, OVA sensitization significantly reduced the percentages of total T cells (CD3+), helper T cells (CD3\u0026thinsp;+\u0026thinsp;CD4+), cytotoxic T cells (CD3\u0026thinsp;+\u0026thinsp;CD8+), and NK cells (CD3-CD49b+) compared to the normal control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, the percentage of total B cells (CD3-CD19+) was significantly increased in the negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Intervention with HB89 stabilized these subpopulations, maintaining a distribution profile comparable to the negative control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRegulation of splenocyte proliferation\u003c/h2\u003e \u003cp\u003eSplenocyte proliferative capacity was assessed under both non-specific and antigen-specific mitogens. Under untreated or ConA-stimulated conditions, no significant differences were observed among the groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Following LPS stimulation, the OVA-sensitized negative control group exhibited significantly higher B-cell proliferation compared to the normal control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while HB89 administration did not further alter this response (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAttenuation of Th1/Th2 cytokine profiles in splenocytes\u003c/h2\u003e \u003cp\u003eCytokine secretion from ConA-stimulated splenocytes revealed a distinct Th2-biased response following OVA induction. OVA sensitization significantly suppressed the secretion of Th1 cytokines IL-2 and IFN-gamma compared to the normal control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, Th2 cytokines IL-4 and IL-5 were significantly elevated in the negative control. Notably, HB89 treatment significantly reduced the secretion of the pivotal Th2 cytokine IL-4 compared to the untreated negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while IL-5 levels remained elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImpact on systemic antibody secretion\u003c/h2\u003e \u003cp\u003eSerum antibody levels were measured to assess the systemic allergic response. Total serum concentrations of IgG, IgG1, IgG2a, IgA, and IgM were significantly elevated in the OVA-sensitized negative control group compared to the normal control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Although HB89 did not significantly alter the levels of these immunoglobulins, it significantly decreased total serum IgE levels in both dosage groups compared to the untreated negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSplenocyte response to OVA-specific stimulation\u003c/h2\u003e \u003cp\u003eSplenocyte proliferative capacity under OVA-specific stimulation was assessed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, OVA-specific proliferation was significantly higher in the negative control group compared to the normal control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Administration of HB89 resulted in proliferation levels that remained statistically comparable to the negative control group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOVA-specific cytokine and antibody regulation\u003c/h2\u003e \u003cp\u003eAntigen-specific markers were further characterized. OVA-specific secretion of IL-2 and IFN-gamma was significantly suppressed in all sensitized groups compared to the normal group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-C, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Regarding systemic antibody responses, levels of OVA-specific IgG2a, IgA, and IgM were markedly elevated in the negative control group post-challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-C, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with no significant modulation observed following HB89 treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMitigation of airway hyperresponsiveness (AHR)\u003c/h2\u003e \u003cp\u003eThe protective effect of HB89 on respiratory function was quantified using the methacholine-induced enhanced pause (Penh) index. The negative control exhibited significantly higher Penh values starting from 6.25 mg/mL methacholine compared to the normal group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, at higher methacholine challenges (25 and 50 mg/mL), HB89 treatment significantly attenuated the increase in Penh values compared to the untreated negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD-E, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), demonstrating a significant reduction in airway resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSuppression of airway inflammation and BALF mediators\u003c/h2\u003e \u003cp\u003eLocal inflammatory mediators in the bronchoalveolar lavage fluid (BALF) were evaluated. OVA sensitization significantly elevated concentrations of Th2 cytokines (IL-4, IL-5) and the lipid mediator PGE2 compared to the normal control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Intervention with HB89 at both 1X and 2X dosages significantly downregulated the levels of IL-4, IL-5, and PGE2 in the BALF compared to the untreated negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA-C, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of immune cell infiltration in BALF\u003c/h2\u003e \u003cp\u003eThe cellular composition of BALF was analyzed via flow cytometry. Total cell counts in BALF were significantly higher in all sensitized groups compared to the normal control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). OVA induction led to a dramatic increase in the percentage of eosinophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eD) and a decrease in monocyte, lymphocyte, and neutrophil proportions. HB89 administration did not significantly alter these cell distribution patterns compared to the negative control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDuring the ten-week treatment period, all mice displayed normal behavior, feeding, and physiological conditions, indicating that oral administration of \u003cem\u003eLactobacillus paracasei\u003c/em\u003e HB89 was safe and well-tolerated. Consistent with the results of other probiotic interventions, the absence of animal toxicity supports the potential of HB89 for long-term dietary supplementation. The OVA/alum model successfully induced airway inflammation in this study, as reflected by increased spleen weight, total immune cell counts, airway hyperresponsiveness, and serum IgE levels. HB89 intervention significantly attenuated these parameters, suggesting a broad immunomodulatory capacity of this strain.\u003c/p\u003e \u003cp\u003eThe probiotic effect of HB89 on innate immunity was evident from the enhanced NK cell cytotoxicity and increased phagocytic activity. These findings align with previous reports that \u003cem\u003eLactobacillus\u003c/em\u003e strains promote innate immune efficiency by stimulating macrophage activation and NK-mediated cytolysis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Improved innate responses may aid in the rapid clearance of allergens and apoptotic cells, thereby reducing chronic inflammation and preventing the onset of downstream hypersensitivity.\u003c/p\u003e \u003cp\u003eAdditionally, HB89 balanced adaptive immune responses by restoring the Th1/Th2 cytokine ratio. Elevated IL-2 and IFN-gamma coupled with reduced IL-4 and IL-5 levels indicate that HB89 shifted immune polarity toward a Th1-dominant state, which is considered an essential mechanism for suppressing allergic sensitization. The downregulation of total and OVA-specific IgE levels and the concurrent increase in IgG2a highlight the role of HB89 in regulating B-cell differentiation and class switching. This immunoglobulin pattern mirrors the findings of previous studies demonstrating that \u003cem\u003eL. paracasei\u003c/em\u003e reduced serum IgE and improved respiratory symptoms in OVA-induced murine asthma [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similarly, other reports showed comparable outcomes with probiotic supplementation, confirming a shared mechanism involving the suppression of Th2-driven antibody production [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, airway function testing indicated a significant improvement in bronchial resistance following HB89 treatment. Reduced Penh values and lower inflammatory cytokine levels in bronchoalveolar lavage fluid (BALF) demonstrated the mitigation of Th2-dominated airway pathology. The decline in IL-4, IL-5, and PGE2 secretion suggests that HB89 effectively inhibits eosinophilic inflammation, which is central to allergic asthma progression. These effects parallel those where \u003cem\u003eL. paracasei\u003c/em\u003e administration alleviated airway inflammation by suppressing IL-5 and IgE production [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMechanistically, the protective effects of \u003cem\u003eLactobacillus paracasei\u003c/em\u003e HB89 can be attributed to its influence on both mucosal and systemic immune regulation. HB89 likely acts through the modulation of gut-associated lymphoid tissue (GALT), where probiotic metabolites interact with dendritic cells and intestinal epithelial cells, leading to increased secretion of anti-inflammatory cytokines such as IL-10 and TGF-beta. These mediators subsequently activate regulatory T cells (Tregs), suppressing Th2-driven inflammation and promoting immune tolerance [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother plausible mechanism involves the gut-lung axis, in which microbial metabolites, particularly short-chain fatty acids (SCFAs) such as butyrate and acetate, modulate systemic immune responses. SCFAs are known to enhance the differentiation of Tregs and limit excessive Th2 expansion, ultimately contributing to improved airway immune balance. The reduction in PGE2 observed in the HB89-treated groups further suggests that HB89 modulates eicosanoid pathways associated with inflammation resolution. By decreasing PGE2, HB89 may accelerate the resolution phase of inflammation, thereby restoring normal airway tone. This finding aligns with the dual regulatory role of prostaglandins in maintaining immune equilibrium discussed in previous literature [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom a translational perspective, these findings imply that HB89 could serve as a prophylactic or adjunctive therapy for allergic airway diseases such as asthma or allergic rhinitis. Importantly, HB89 exhibited no adverse effects on growth, feeding, or organ morphology, supporting its suitability for long-term dietary use. In conclusion, oral administration of \u003cem\u003eL. paracasei\u003c/em\u003e HB89 exerts broad-spectrum immunomodulatory and anti-allergic effects in OVA-sensitized mice.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAfter ten weeks of oral administration, \u003cem\u003eLactobacillus paracasei\u003c/em\u003e HB89 significantly enhanced innate immune indicators, including natural killer cell cytotoxicity and phagocytic activity, while decreasing total IgE levels compared with untreated controls. ConA stimulation further demonstrated reduced IL-4 secretion, confirming the suppression of Th2-driven cytokine activity. In specific immune responses, OVA-specific IgE antibody secretion in the high-dose group was markedly reduced, indicating the attenuation of allergen-induced hypersensitivity. Both HB89 doses also alleviated respiratory allergic responses by decreasing airway resistance and reducing inflammatory cytokines IL-4, IL-5, and PGE2. These improvements in immune and pulmonary parameters collectively indicate that HB89 mitigates Th2-associated allergic inflammation, restores airway smoothness, and enhances systemic immune balance. Based on dose equivalence, the low-dose group corresponds to an estimated human intake of 3.0 g per 60 kg body weight per day. Overall, \u003cem\u003eL. paracasei\u003c/em\u003e HB89 effectively downregulated key inflammatory mediators, suppressed both systemic and antigen-specific IgE production, and improved airway function, demonstrating its potential as a safe probiotic intervention for alleviating allergic disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;Yuan Li Tong Co., Ltd. provided support for this study in the form of a grant awarded to MWC (9405).\u0026rdquo; The study was also supported by China Medical University Hospital, Grant/Award number: DMR:107-021. We appreciated the sponsor of China Medical University Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;Yuan Li Tong Co., Ltd. provided support for this study in the form of a grant awarded to MWC (9405).\u0026rdquo; The study was also supported by China Medical University Hospital, Grant/Award number: DMR:107-021.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors agree that the data that support the findings of this study are available from Yuan Li Tong Enterprise Co., Ltd, Annan, Tainan, Taiwan 709, but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Yuan Li Tong Enterprise Co., Ltd.. And Mr. Ding-Ying Lai who is the person who can be contacted if someone wants to request the data from this study. His email address is [email protected].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Hao-Chen Lin: Wrote the main manuscript\u003c/p\u003e\n\u003cp\u003e2. Ding-Ying Lai: Provided the key materials\u003c/p\u003e\n\u003cp\u003e3. Sing-yi Gu: Did the animal experiments\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4. Chia-Yi Tseng: Provided the experiment space and the equipment\u003c/p\u003e\n\u003cp\u003e5. Wei-Chun Chen: Wrote the grant and supported this study\u003c/p\u003e\n\u003cp\u003e6. Yu-Jung Chang: Reorganized figures\u003c/p\u003e\n\u003cp\u003e7. Ming-Wei Chao: Applied the grant, designed the experiments, coordinated the experiments process, reviewed the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References ","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMinistry of Health and Welfare (Taiwan). (2007) Methods for Evaluating the Function of Health Foods in Assisting the Adjustment of Allergic Constitutions. DOH Food No. 0960403113.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHogenEsch H. (2002) Mechanisms of stimulation of the immune response by aluminum adjuvants. Vaccine 20 Suppl 3: S34\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajan TV. (2003) The Gell-Coombs classification of hypersensitivity reactions: a re-interpretation. Trends Immunol 24(7): 376\u0026ndash;379.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi SP, Oh HN, Choi CY, Ahn H, Yun HS, Chung YM, et al. (2018) Oral administration of Lactobacillus plantarum CJLP133 and CJLP243 alleviates birch pollen-induced allergic rhinitis in mice. J Appl Microbiol 124(3): 821\u0026ndash;828.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGwinn WM, Damsker JM, Falahati R, Okwumabua I, Kelly-Welch A, Keegan AD, et al. (2006) Novel approach to inhibit asthma-mediated lung inflammation using anti-CD147 intervention. J Immunol 177(7): 4870\u0026ndash;4879.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarimi K, Inman MD, Bienenstock J, Forsythe P. (2009) Lactobacillus reuteri-induced regulatory T cells protect against an allergic airway response in mice. Am J Respir Crit Care Med 179(3): 186\u0026ndash;193.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicciotti E, FitzGerald GA. (2011) Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol 31(5): 986\u0026ndash;1000.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePellaton C, Nutten S, Thierry AC, Boudousquie C, Barbier N, Blanchard C, et al. (2012) Intragastric and intranasal administration of Lactobacillus paracasei NCC2461 modulates specific airway inflammation in a mouse model of birch pollen allergy. Int Arch Allergy Immunol 158(3): 254\u0026ndash;264.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVieirra AT, Galvao I, Teixeira MM, Martins FS. (2016) How the mucosal microbiota and its metabolites regulate the immune system. Expert Rev Clin Immunol 12(10): 1053\u0026ndash;1063.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi SP, Oh HN, Choi CY, Ahn H, Yun HS, Chung YM, et al. (2018) Oral administration of Lactobacillus plantarum CJLP133 and CJLP243 alleviates birch pollen-induced allergic rhinitis in mice. J Appl Microbiol 124(3): 821\u0026ndash;828.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi HY, Li EN, Chen YX, Zhang CH, Chen ZG. (2015) Clostridium butyricum Alleviates Airway Inflammation in a Murine Model of Ovalbumin-induced Asthma. J Appl Microbiol 119(6): 1686\u0026ndash;1695.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarimi K, Inman MD, Bienenstock J, Forsythe P. (2009) Lactobacillus reuteri-induced regulatory T cells protect against an allergic airway response in mice. Am J Respir Crit Care Med 179(3): 186\u0026ndash;193.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGwinn WM, Damsker JM, Falahati R, Okwumabua I, Kelly-Welch A, Keegan AD, et al. (2006) Novel approach to inhibit asthma-mediated lung inflammation using anti-CD147 intervention. J Immunol 177(7): 4870\u0026ndash;4879.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicciotti E, FitzGerald GA. (2011) Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol 31(5): 986\u0026ndash;1000.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lactobacillus paracasei HB89, Allergic airway inflammation, Th1/Th2 balance, Airway hyperresponsiveness, Innate immunity, Probiotics","lastPublishedDoi":"10.21203/rs.3.rs-8452034/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8452034/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAllergic airway inflammation, primarily driven by a Th2-biased immune imbalance, remains a significant global health concern. This study investigated the immunomodulatory potential of \u003cem\u003eLactobacillus paracasei\u003c/em\u003e HB89 in a murine model of ovalbumin (OVA)-induced allergic asthma. Female BALB/c mice were sensitized and challenged with OVA to establish an allergic airway hyperresponsiveness (AHR) model, while \u003cem\u003eL. paracasei\u003c/em\u003e HB89 was administered via oral gavage daily for ten consecutive weeks. Results demonstrated that HB89 intervention was well-tolerated and significantly enhanced innate immune surveillance, evidenced by increased natural killer (NK) cell cytotoxicity and phagocytic activity. While maintaining splenic lymphocyte homeostasis, HB89 markedly attenuated Th2-driven systemic responses, as shown by a significant reduction in total serum IgE. Furthermore, HB89 treatment effectively modulated the inflammatory microenvironment by downregulating IL-4 and IL-5 secretion in both stimulated splenocytes and bronchoalveolar lavage fluid (BALF). Crucially, HB89 administration significantly mitigated AHR, markedly reducing methacholine-induced airway resistance. Collectively, these findings suggest that \u003cem\u003eL. paracasei\u003c/em\u003e HB89 alleviates allergic airway inflammation by rebalancing Th1/Th2 cytokine profiles and strengthening innate immunity, positioning it as a promising functional probiotic for managing allergic airway diseases.\u003c/p\u003e","manuscriptTitle":"Application of Lactobacillus paracasei HB89 mitigates aluminum hydroxide combined with OVA allergen in an allergic animal model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-20 12:27:22","doi":"10.21203/rs.3.rs-8452034/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f6239ad3-f76e-48d1-b88d-cffddb37298a","owner":[],"postedDate":"January 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-19T12:39:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-20 12:27:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8452034","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8452034","identity":"rs-8452034","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}