Heat-Treated Lacticaseibacillus rhamnosus Strains Modulate Inflammatory and Metabolic Processes in In Vitro Systems Relevant to Canine Osteoarthritis

Abstract

Highlights

What are the main findings?

• Heat-treated Lacticaseibacillus rhamnosus strains (PRIOME^® JH and HT-PB01) exhibited anti-inflammatory activity and type II collagen synthesis in cultured canine chondrocytes.

• In Caenorhabditis elegans models, both strains mitigate intestinal permeability, reduce fat accumulation, and enhance muscle structure and functionality, suggesting their potential to reduce risk factors associated with osteoarthritis.

What are the implications of the main findings?

• These results identify PRIOME^® JH and HT-PB01 as promising postbiotic candidates for further investigation regarding their potential benefit for canine joint health.

• This study provides a preclinical basis for future in vivo investigation aimed at evaluating postbiotic strategies to address multiple risk factors associated with canine osteoarthritis.

Abstract

Canine osteoarthritis is a progressive degenerative joint condition characterized by inflammation, structural damage, pain, and impaired joint function. Osteoarthritis risk is influenced not only by well-established factors such as age, genetics, obesity and underlying arthropathies, but also by systemic conditions such as low-grade inflammation and sarcopenia. Current interventions are primarily focused on pain relief rather than mitigating physiological impairment, despite recent veterinary guidelines. In this study, several preclinical models were employed to identify postbiotic candidates with potential to promote canine joint health. An initial screening of nine heat-treated bacterial strains identified two Lacticaseibacillus rhamnosus strains, PRIOME^® JH and HT-PB01, which demonstrated significant anti-inflammatory activity on canine chondrocytes. Further characterization revealed that these two strains significantly increased type II collagen synthesis in canine chondrocytes and modulate immune responses in canine macrophages, enhancing Interleukin-10 secretion over Interleukin-12. Additionally, both strains showed potential in mitigating risk factors such as gut inflammation and permeability, fat accumulation and sarcopenia in Caenorhabditis elegans models. In conclusion, this study identified two promising postbiotic candidates, PRIOME^® JH and HT-PB01, with the potential to target key risk factors associated with osteoarthritis through multifactorial mechanisms. These findings emphasize conducting dog clinical trials to evaluate their relevance in osteoarthritis.

1. Introduction

Canine osteoarthritis (OA) is a non-autoimmune degenerative condition, caused by progressive inflammation and structural changes of the joints associated with pain, stiffness disability, and poor joint function, which ultimately result in a deterioration in the quality of life of affected dogs [1,2]. OA development is related to high concentrations of proinflammatory mediators such as Interleukin (IL)-1 beta (β), Tumor Necrosis Factor Alpha (TNF-α), or IL-6 in the cartilage and synovial area [3]. The accumulation of proinflammatory signals in the joints may activate resident immune cells, such as macrophages [4], and stimulate the release of proteolytic enzymes, such as matrix metalloproteinases (MMPs), which play a critical role in cartilage degradation [3,4]. Among the key molecules targeted by MMPs is type II collagen, a principal component of the cartilage extracellular matrix. Degradation and loss of type II collagen critically impair joint functionality. Type II collagen is the primary constituent of the cartilage matrix. Beyond its structural role, it also acts as a signaling molecule that regulates chondrocytes and cartilage homeostasis [5]. A reduction in type II collagen levels disrupts chondrocyte homeostasis, promoting their transition toward a hypertrophic state. Hypertrophic chondrocytes exhibit reduced synthesis of type II collagen, thereby accelerating cartilage matrix degradation. This self-perpetuating cycle ultimately results in cartilage ossification, a process that irreversibly impairs joint functionality [5].

Although OA is a localized condition in joints, multiple studies show that its early development is influenced by several systemic factors [3,6,7,8,9]. Among these, obesity is a well-established contributor, associated with the initial stage of OA pathogenesis. Locally, the accumulation of adipocytes in various body sites, particularly within the knee (e.g., the infrapatellar fat pad), acts as an endocrine organ that secretes adipokines. These molecules play a pivotal role in modulating inflammatory processes and contribute to the local inflammation of canine cartilage [10]. At systemic levels, obesity has also been associated with increased gut permeability in dogs [9,11]. This impaired intestinal barrier function contributes to low-grade inflammation due to the translocation of bacterial endotoxins from the lumen. Bacterial lipopolysaccharides (LPS) represent a detrimental factor in OA development. Beyond triggering the innate immune response and low-grade inflammation, they also stimulate macrophage activation, resulting in increased secretion of proinflammatory cytokines related to OA pathogenesis [12]. A recent cross-sectional study, involving almost 12,000 human participants, identified sarcopenia as an additional risk factor for OA development [13]. The study suggests that reduced muscle functionality may compromise joint stability, thereby accelerating cartilage degradation. A similar effect of sarcopenia in OA progression has been observed in rat models, as reported by Xu et al., 2020 [14].

Current pet care strategies focus on alleviating pain and improving mobility associated with OA. Non-steroidal anti-inflammatory drugs (NSAIDs), together with weight management, exercises, and physiotherapy, represent the primary approach for managing pain in canine OA. However, the long-term use of NSAIDs may lead to various side effects, including gastrointestinal and renal complications [15]. Furthermore, NSAIDs do not prevent OA progression and the associated joint degradation. Even the use of supplements, such as chondroitin sulfate, glucosamine, and omega-3, represent common strategies during OA stages associated with pain [16,17]. However, their use is not unanimously recommended when OA risk factors are detected in dogs, particularly in the absence of clinical symptoms. According to experts, if a dog maintains a balanced diet, the decision to avoid supplementation is primarily influenced by factors such as the additional cost, the difficulty of sourcing high-quality supplements free from unwanted ingredients, the risk of obesity, and the challenges of administering an appropriate dosage [17]. Therefore, in 2023, an international panel of experts developed guidelines intended to serve as a practical reference for veterinarians in the management of canine OA. This framework emphasizes the importance of preventive strategies, advocating for a proactive and continuous approach to delaying or mitigating the OA progression [17].

Microbiome-based solutions, including probiotics or postbiotics, are emerging as a promising new strategy to support joint health, as they can mitigate the risk factors associated with OA. In this context, postbiotics, the preparation of inanimate microorganisms and/or their components that confer a health benefit on the host [18], are particularly interesting in animal nutrition and pet care. They offer a stable alternative to live probiotics, which can be sensitive to environmental conditions such as temperature and pH, and therefore to the industrial processes and storage conditions of pet supplements.

Several studies have shown that postbiotics derived from lactic acid bacteria and bifidobacteria exert beneficial health effects, including anti-inflammatory properties [19,20,21] and fat-reduction capabilities [22,23]. In the specific case of OA, a postbiotic demonstrated promising effects in guinea pigs, reducing cartilage degradation and the loss of type II collagen, when supplemented with a lyophilized inactivated culture of Bifidobacterium longum CBi0703 [24]. To date, to the best of our knowledge, no clinical trials have evaluated probiotics or postbiotics interventions for OA in dogs. Nevertheless, several clinical trials in humans have highlighted the pain-relieving potential of probiotic supplementation. For instance, Lacticaseibacillus casei Shirota, Latilactobacillus sakei LB-P12, and L. rhamnosus PB01 have been associated with reduced pain in individuals with OA [25,26,27]. These findings are consistent with those of a human clinical trial, in which L. rhamnosus PB01 supplementation significantly increased pressure pain thresholds in individuals with knee OA. Additionally, 57% of the patients who received the probiotic reported improvements in overall health and well-being, as indicated by patient-reported outcomes [25]. L. rhamnosus PB01 has also shown promising results in animal models, particularly in mice, where it produced significant weight reduction and a notable decrease in pain sensitivity [28]. In the present study, we characterized different heat-treated (HT) bacterial strains using a canine chondrocyte-based model to evaluate their potential anti-inflammatory activity. Based on this initial screening, complementary preclinical assessments were performed with the most promising HT strains, Lacticaseibacillus rhamnosus PRIOME^® JH and HT-PB01. These strains were further evaluated and assessed in canine chondrocyte cells to examine their effects on type II collagen production, and in Caenorhabditis elegans (C. elegans) to investigate their influence on fat accumulation, intestinal permeability, and muscle cell structure and functionality.

2. Materials and Methods

2.1. Bacterial Strains and Culture Conditions

Nine strains belonging to lactic acid bacteria genera and Bifidobacterium (Table 1) were cultured overnight at 37 °C in Man–Rogosa–Sharpe (MRS) (Scharlab, Sentmenat, Spain) broth medium supplemented with 0.05% (w/v) cysteine (Coralim aditivos SL, Paterna, Spain) in anaerobic conditions for 24 h. To prepare the HT strains, bacterial cultures were harvested by centrifugation and washed with NaCl solution (0.9%). Afterwards, cells were heat-treated by autoclaving at 121 °C for 20 min, quantified using Cytoflex flow cytometer (Beckman Coulter, Suzhou, China), and used to prepare the different doses for each experiment.

Table 1.

Collection of potential candidates evaluated during the screening.

Bacterial Strains	Abbreviation
Bifidobacterium animalis subsp. lactis A	HT-B. lactis A
Lacticaseibacillus rhamnosus A	HT-L. rhamnosus A
Lacticaseibacillus rhamnosus B	HT-L. rhamnosus B
Lacticaseibacillus rhamnosus PB01 DSM 14870	HT-PB01
Lacticaseibacillus rhamnosus NCIMB 30446	PRIOME® JH
Limosilactobacillus reuteri A	HT-L. reuteri A
Lacticaseibacillus paracasei A	HT-L. paracasei A
Lacticaseibacillus paracasei B	HT-L. paracasei B
Lacticaseibacillus paracasei C	HT-L. paracasei C

2.2. Cell Culture Maintenance

Primary chondrocytes (Cn402-05) isolated from healthy canine joint tissue were obtained from Cell Applications Inc. (San Diego, CA, USA). Cells were routinely maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin-Streptomycin (100 U/mL–100 µg/mL).

Human colonic epithelial cell line HT-29 was obtained from the American Type Culture Collection (ATCC) No. HTB-38. Cells were cultured in McCoy’s 5A medium supplemented with 10% FBS and antibiotics.

Canine macrophage cell line DH82 was obtained from the ATCC (ATCC no. CRL-3590™). Cells were routinely maintained in DMEM medium supplemented with 10% FBS, 2 mM L-Glutamine, 1% non-essential amino acids, and Penicillin-Streptomycin (100 U/mL–100 µg/mL).

All cells were grown at 37 °C in a humidified atmosphere with 5% CO₂. Otherwise stated, all cell culture reagents were obtained from Thermo Fisher Scientific (Madrid, Spain). The catalog numbers of the reagents used in this section and throughout this study are listed in Supplementary Table S1.

2.3. Anti-Inflammatory Activity in Canine Chondrocytes

For the anti-inflammatory assays with canine chondrocytes, cells were seeded in 96-well plates (1 × 10⁴ cells/well) for 24 h. Afterwards, cells were challenged with canine IL-1β (Kingfisher Biotech, Saint Paul, MN, USA; 10 ng/mL) in the presence or absence of 9 HT strains (10⁷ cells/mL) for 16 h at 37 °C individually. Then, cell supernatants from each condition were harvested and stored at −20 °C until IL-6 determination by Luminex™ technology. ProcartaPlex™ Mix and Match commercial kit (Thermo Fisher Scientific, Madrid, Spain) was used according to the manufacturer’s instructions. Data are reported as the percentage of IL-6 secretion compared with cells stimulated with IL-1β in the absence of HT strains. Cell viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg/mL; Merck, Madrid, Spain) to rule out inhibition due to cytotoxicity promoted by the HT bacterial cells. Assays were performed in triplicate.

2.4. Type II Collagen Production in Canine Chondrocytes

To evaluate the capacity of inactivated bacterial cells to induce collagen production, canine chondrocytes were encapsulated in alginate beads in 24-well plates and cultured for 21 days according to chondrogenesis kit instructions (Cell Applications Inc., San Diego, CA, USA) and the protocol previously described by De Ceuninck F. et al., 2004 [29].

Once the cells were near confluence, they were first trypsinized and resuspended in sodium alginate solution (1 × 10⁶ cells/mL). Drops were poured using a 22-gauge needle into a CaCl₂ solution in agitation to allow its polymerization. Alginate beads were washed 5 times with NaCl solution (0.9%) and finally, one more time in chondrocyte differentiation medium (Cell Applications Inc., San Diego, CA, USA). A total of 10 alginate beads were seeded per well (approximately 1 × 10⁵ cells). Then, the encapsulated chondrocytes were cultured in chondrocyte differentiation medium alone (control) or in the presence of HT strains (PRIOME^® JH or HT-PB01) at doses of 10⁸ cells/mL for 21 days. Medium was replaced twice a week. The study was performed in three independent assays.

Type II collagen and matrix metalloproteinase (MMP)-1 levels were measured at the final time point under all experimental conditions. For type II collagen quantification, chondrocytes were first recovered by incubating the alginate beads with the depolymerization solution. Subsequently, 3 incubation steps with a pepsin digestion (0.1 mg/mL, Merck) for 24 h, followed by elastase digestion (0.1 mg/mL, Merck) for 24 h, were carried out to solubilize type II collagen synthesized by the cells. Both proteins were quantified using ELISA assay kits according to the manufacturer’s instructions (Chondrex Inc, Woodinville, WA, USA; and R&D Systems, Minneapolis, MN, USA). Data were normalized to the total protein content of the samples, measured by the Pierce™ bicinchoninic acid (BCA) method (Thermo Fisher Scientific).

2.5. Immunomodulatory Effect in Canine Macrophages

DH82 macrophages were seeded at 1.0 × 10⁵ cells in 96-well plates. The next day, cells were stimulated with postbiotics at 10⁸ cells/mL in DMEM medium without antibiotics for 5 h at 37 °C. Subsequently, supernatants were collected and stored at −20 °C until cytokine analysis. Cytokine quantitation was carried out using the Luminex 200™ System with a 2-cytokine canine panel (IL-10 and IL-12) from Thermo Fisher following the manufacturer’s instructions. The assay was performed three times independently.

2.6. Anti-Inflammatory Effects on Gut Cells

Due to the lack of commercially available dog intestinal cell lines, anti-inflammatory activity of HT bacteria was assessed in HT-29 cells, derived from human colorectal adenocarcinoma. Cells were seeded in 96-well plates (5 × 10⁴ cells/well) and cultured for 6 days. Then, cells were treated with 2 ng/mL of TNF-α (Merck) in the presence or absence of the HT bacteria (10⁹ cells/mL) for 3 h at 37 °C. Cell supernatants of each condition were harvested and stored at −20 °C until IL-8 determination by Luminex technology using a ProcartaPlex™ Mix and Match commercial kit (Thermo Fisher Scientific). Viability of HT-29 cells was checked by the MTT assay as described above. Experiments were performed in triplicate.

2.7. C. elegans Strains and Maintenance Conditions

C. elegans strains N2 Bristol (wild-type) and DM8005 (raIs5 [myo-3p::GFP::myo-3 + rol-6(su1006)]) were obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota (Minneapolis, MN, USA) and maintained at 20 °C on Nematode growth Medium (NGM) plates with Escherichia coli strain OP50 as normal diet for nematodes [30].

2.8. Gut Barrier Permeability in C. elegans

To evaluate intestinal permeability, age-synchronized worms of wild-type strain N2 were maintained in NGM or NGM supplemented with the corresponding HT strain (PRIOME^® JH or HT-PB01) at a dose of 10⁸ cells/plate, and incubated at 20 °C. All plates were also seeded with OP50 E. coli. To induce intestinal permeability, L4-nematodes were exposed to 0.5 µg/mL methotrexate (MTX; Merck; St Louis, MO, USA) for 24 h. A condition without intestinal cell damage was included (NGM control medium). Evaluation of the nematode’s intestinal barrier integrity was performed by Nile red staining (0.05 µg/mL) (Merck; St Louis, MO, USA). Nematodes in each condition were examined under a fluorescence stereomicroscope Nikon-SMZ18 (Nikon, Tokyo, Japan) to detect the infiltration of the dye into the internal cavity of the worm as a consequence of intestinal damage. Results are shown as the percentage of fluorescence in each treatment compared to worms without treatment (control). Experiments were performed in triplicate.

2.9. Fat-Reducing Effect in C. elegans

Age-synchronized worms of the wild-type N2 strain were cultured in NGM + OP50 (control medium) or NGM + OP50 supplemented with HT strains (10⁸ cells/plate). The fat content in worms was measured by the Nile red staining method, following the protocol previously described by Martorell et al., 2012 [31]. Orlistat (6 µg/mL) (anti-obesity drug) was included as positive control of the assay. Worms’ population was incubated until reaching the young adult stage. After this period, fluorescence of the nematodes was measured using a FP-8300 system (JASCO Analytical Instrument, Easton, MD, USA). Assays were performed in triplicate. Results are shown as the percentage of fluorescence in each treatment with respect to NGM + OP50 without supplement (control).

2.10. Muscle Structure Protection in C. elegans

C. elegans muscle structure was analyzed by fluorescence microscopy using the DM8005 transgenic strain containing a GFP-tagged myo-3 protein. DM8005 worms were synchronized in NGM plates supplemented with E. coli OP50 as food and with PRIOME^® JH or HT-PB01 strains (10⁸ cells/plate). Animals were transferred onto fresh plates every 2 days after the young adult stage to remove progeny and prevent population starvation. To image muscle structure, at day 11 of adulthood, 40–50 animals per condition were picked onto a microscope slide containing 4% agarose and imaged immediately using a fluorescence microscope Zeiss Axio Imager M2 (Zeiss, Jena, Germany) at 20× magnification. Image analysis was conducted manually by a trained observer. The general structure and organization of worm myofilaments were categorized, considering irregularities such as gaps and non-parallel alignment of myofilaments. These observations were used to classify the entire worm into the corresponding group based on myofilament structure (well-organized, moderately disorganized, or severely disorganized). Experiments were performed in triplicate.

2.11. Evaluation of Muscle Functionality

The C. elegans N2 strain was cultured to the L4 larval stage as previously described in NGM plates seeded with OP50, with or without supplementation of PRIOME^® JH or HT-PB01 postbiotic strain (dose of 10⁸ cells/plate). Upon reaching the L4 stage, worms were treated with 5-Fluoro-2′-deoxyuridine to prevent the emergence of new progeny and subsequently transferred to fresh plates containing OP50 and supplemented with each HT strain (n = 600 worms per condition, performed in three independent experiments). The worms were then introduced into the miniTower, an automated system to track nematode mobility, and maintained at 20 °C for 7 days. Worms’ mobility was assessed twice daily using automated image analysis provided by the miniTower system. Mobility data were expressed as a percentage relative to the control condition on day 1, and mobility curves were generated for each condition.

2.12. Statistical Analyses

Results are expressed as the mean ± standard deviation (SD). Data normality was first assessed using the Shapiro–Wilk/Kolmogorov–Smirnov tests. Depending on whether the data followed a normal distribution, the appropriate parametric or non-parametric statistical tests were applied. In addition, prior to the parametric tests, a Brown–Forsythe test was applied to verify no variance differences. For the anti-inflammatory effect in canine chondrocyte cells and fat-reduction effect in C. elegans, data were analyzed using one-way ANOVA with Dunnett’s post-hoc comparison test. For the anti-inflammatory effect in HT-29 cells, Welch’s ANOVA test with Dunnett’s post-hoc comparison test was applied. For type II collagen production and gut barrier permeability data in C. elegans, a Kruskal–Wallis test followed by Dunn’s multiple comparison test was used. For the immunomodulation study in DH82 macrophages, a Mann–Whitney test was applied, and the muscle structure protection study in C. elegans was analyzed using a Chi-square test. Finally, to compare the mobility of different conditions, a mixed-effects analysis was performed. All the analyses were performed with GraphPad Prism 9 software (GraphPad Software; Boston, MA, USA). A p-value lower than 0.05 was considered statistically significant.

3. Results

3.1. Anti-Inflammatory Effect of Postbiotics in Canine Chondrocyte Cells

To evaluate the anti-inflammatory potential of postbiotics, a canine chondrocyte model was used. In the initial screening, nine different postbiotics were evaluated. Cells were challenged with recombinant canine IL-1β in the presence or absence of the collection of postbiotics, and IL-6 levels were analyzed. IL-1β was selected as this cytokine plays an important role in the early steps of osteoarthritis [32,33]. In particular, IL-1β is overexpressed in the damaged articular joint, which triggers the secretion of a wide variety of cytokines/chemokines, including IL-6 [4]. As shown in Figure 1 and Supplemental Figure S1, the proinflammatory stimulus with IL-1β increased IL-6 release in canine chondrocyte primary cells (p-value < 0.0001 vs. negative control). For each assay, the percentage of IL-6 secretion was calculated with respect to the IL-6 secretion of cells stimulated with IL-1β without HT strains. Among the HT strains tested, PRIOME^® JH and HT-PB01 significantly reduced IL-6 release in IL-1β-stimulated cells compared with cells stimulated without a HT strain (22.20 ± 7.20% and 23.53 ± 8.83%, respectively). The reduction in IL-6 secretion by PRIOME^® JH and HT-PB01 was statistically significant, with a p-value of 0.0053 for PRIOME^® JH and 0.0032 for HT-PB01. Furthermore, subsequent MTT evaluation confirmed that the addition of postbiotics did not affect the cells’ viability (p-value > 0.05, Figure S2). No significant reduction in IL-6 was observed for the other HT strains tested (p-value > 0.05 vs. IL-1β).

Figure 1.

Screening of the anti-inflammatory effect of different heat-treated strains in canine chondrocytes. IL-6 secretion by primary canine chondrocytes stimulated with IL-1β was measured in the presence of a collection of candidates. Data are represented as the percentage of IL-6 secretion relative to IL-1β stimulated condition without inactivated cells. Control condition refers to chondrocytes stimulated with IL-1β, whereas negative control (NC) corresponds to chondrocytes without stimulation. Data are presented as the mean ± SD of three independent experiments. Statistical test: One-way ANOVA with Dunnett’s multiple comparisons test (Postbiotic vs. Control). Significance levels were indicated as follows: p-value > 0.05 (ns: non-significant), p-value < 0.01(**), p-value < 0.0001 (****).

3.2. HT Strains Induced Insoluble Type II Collagen Production in Canine Chondrocytes

Given the critical role of type II collagen in joint health, the effects of the most promising postbiotic candidates, PRIOME^® JH and HT-PB01, on insoluble type II collagen production were evaluated in canine chondrocytes. As shown in Figure 2, chondrocytes cultured in the presence of PRIOME^® JH exhibited a significant increase in type II collagen production, reaching 405.23 ± 53.62% compared to the untreated control (p-value = 0.03). Similarly, HT-PB01 stimulated type II collagen production, with levels reaching 367.7 ± 51.16% relative to the control condition; however, these results were not significant (p-value = 0.19). In addition, postbiotic treatment did not affect MMP-1 secretion levels in the canine chondrocytes (p-value > 0.05; Supplemental Figure S3). These results highlight the capacity of both HT strains to enhance type II collagen production in canine chondrocytes, suggesting their potential for promoting joint health.

Figure 2.

Insoluble type II collagen production by primary canine chondrocytes cultured in spheroids structure. Data are presented as the mean ± SD of three independent experiments and expressed as the percentage of insoluble type II collagen production relative to the control condition. In the control condition, canine chondrocytes were cultured without supplementation of heat-treated strains. Statistical test: Kruskal-Wallis test, followed by Dunn’s multiple comparison test (Control vs. Postbiotic). Significance levels were indicated as follows: p-value < 0.05 (*).

3.3. Immunomodulatory Effect of HT Strains in Canine Macrophages

To assess the interaction between HT strains and the immune system, PRIOME^® JH and HT-PB01 were co-cultured with canine macrophages. As illustrated in Figure 3, the addition of HT strains induced the differential secretion of IL-10 and IL-12 compared to unstimulated macrophages, which produced only minimal amounts of IL-10 (3.71 ± 1.64 pg/mL). Specifically, PRIOME^® JH stimulated IL-10 secretion to 222.22 ± 100.17 pg/mL, while IL-12 secretion reached 61.55 ± 22.54 pg/mL. Similarly, HT-PB01 demonstrated immunomodulatory effects on canine macrophages, increasing IL-10 secretion to 145.88 ± 72.78 pg/mL and IL-12 secretion to 73.64 ± 19.69 pg/mL.

Figure 3.

Cytokine secretion (IL-10 and IL-12) of DH82 macrophages challenged with PRIOME^® JH or HT-PB01. Data are expressed as mean ± SD of at least three independent experiments. Statistical test: Mann–Whitney test was applied to compare cytokine secretion in each condition. Significance levels were indicated as follows: p-value < 0.01 (**).

Both postbiotics significantly enhanced IL-10 production compared to IL-12 (p-value = 0.0017 and p-value = 0.0040, respectively). The calculation of the IL-10/IL-12 secretion ratio highlights the immunomodulatory effects of both postbiotics, promoting a more anti-inflammatory state characterized by high IL-10/IL-12 ratios. Cells supplemented with PRIOME^® JH exhibited an IL-10/IL-12 ratio of 3.42 ± 0.74, while cells treated with HT-PB01 displayed a ratio of 1.89 ± 0.5.

3.4. Anti-Inflammatory Effect of HT Strains in HT-29 Cells

The effects of the HT strains PRIOME^® JH and HT-PB01 on IL-8 release were evaluated in the HT-29 intestinal cell line exposed to a proinflammatory stimulus. HT-29 cells were challenged with TNF-α in the presence or absence of a HT strain. For each assay, the percentage of secretion was calculated relative to the challenged cells with TNF-α. As shown in Figure 4, the proinflammatory stimulus with TNF-α increased IL-8 release in HT-29 cells (p-value < 0.0001 vs. negative control). Both potential postbiotics, PRIOME^® JH and HT-PB01, significantly reduced the secretion of IL-8, 24.24± 14.94% and 46.21 ± 18.10%, respectively, with p-value < 0.0001 vs. TNF-α in the case of HT-PB01, and p-value = 0.0026 vs. TNF-α for PRIOME^® JH.

Figure 4.

Anti-inflammatory effect of different heat-treated strains in the HT-29 cell line. IL-8 secretion by HT-29 epithelial cells upon TNF-α treatment in the presence of PRIOME^® JH and HT-PB01. Data are represented as the percentage of IL-8 secreted by the challenged condition with TNF-α and without supplementation (control). Negative control (NC) was cells without stimulation. Data are expressed as the mean ± SD of three independent experiments. Statistical analysis: Welch’s ANOVA test followed by Dunnett’s post-hoc tests for pairwise comparisons. The levels of significance were denoted as follows: p-value < 0.01 (**), p-value < 0.0001 (****).

3.5. Gut Barrier Integrity Preservation by HT Strains in C. elegans

Due to its experimental simplicity and translational relevance to mammalian systems, a C. elegans model was employed to evaluate the potential protective effect of HT strains on gut barrier integrity. Nematodes were fed with both HT strains, PRIOME^® JH or HT-PB01, and the fluorescence intensity of Nile red was measured after the treatment with methotrexate. Worms supplemented with PRIOME^® JH and HT-PB01 showed a protective effect against gut damage, due to the reduction in gut permeability, as reflected in fluorescence values of 97.6 ± 2.7% and 98.0 ± 3.4%, respectively, compared to the damaged condition, which exhibited a fluorescence value of 120.2 ± 6.6%. Moreover, both HT strains displayed fluorescence intensity comparable to the undamaged control (100 ± 3.7%) (Figure 5A). These findings suggest a protective effect of both postbiotics to intestinal permeability, as observed in the microscopic images for Nile red staining (Figure 5B).

Figure 5.

Protective capabilities of PRIOME^® JH and HT-PB01 in gut permeability using the C. elegans model. (A) Gut permeability protection effect represented by the percentage of fluorescence intensity of Nile red vs. control condition with damage. Gut damage was induced with methotrexate (MTX). Negative control (NC) was worms without damage and supplementation. Data are the mean ± SD of three independent experiments (N = a total of 90 worms per condition). Statistical analysis: Data were analyzed using a Kruskal–Wallis test, followed by Dunn’s multiple comparison test. The levels of significance were represented as: p-value < 0.0001 (****). (B) Representative images of Nile red staining in C. elegans under fluorescence microscopy. 20× magnification. Scale bar = 1000 µm.

3.6. Fat Reduction in C. elegans

To study the potential effect of both HT strains in fat deposition, C. elegans worms were cultured in the presence of the PRIOME^® JH or HT-PB01. As shown in Figure 6, a significant fat-reduction effect was observed in both PRIOME^® JH and HT-PB01 fed conditions (both p-value < 0.0001), with a reduction of 24.2 ± 1.74% and 20.5 ± 4.34%, respectively, compared with worms maintained on a standard diet lacking HT-strain supplementation.

Figure 6.

Fat-reduction properties of PRIOME^® JH and HT-PB01 in the C. elegans model, represented as the percentage of fluorescence of Nile red vs. control with a standard feeding condition. N = 180 worms per condition. Data are represented as the mean ± SD. One-way ANOVA was applied with Dunnett’s comparison test. The levels of significance were denoted as follows: p-value < 0.0001 (****).

3.7. Evaluation of Muscle Support in C. elegans Model

To study the protective effect of postbiotics on muscle cell structure, the myofilament structure of individual 11-day-old worms was analyzed by fluorescence image in the C. elegans DM8005 strain. As shown in Figure 7A and Figure 7B, severe disorganization was observed in 1% and 4% of worms fed with PRIOME^® JH and HT-PB01, respectively, compared to 20% in the control group. In addition, the presence of worms with well-organized muscle structure was higher in postbiotics compared to the control condition, with an increase of 18% and 22% worms for PRIOME^® JH and HT-PB01, respectively. The addition of each HT strain significantly protected the age-related muscle structure disorganization in comparison to the control-fed condition (p-value < 0.0001).

Figure 7.

Muscle structure protection properties of PRIOME^® JH and HT-PB01. Microscopic analysis of the myofilaments in C. elegans DM8005 reporter strain was undertaken. (A) Percentage of worms showing each type of myofilament organization (well-organized, moderately disorganized, or severely disorganized) fed with or without PRIOME^® JH or HT-PB01. Between 75–80 worms were used per condition. Chi-square test was applied for the statistical test, where p-value < 0.0001 was represented by ****. (B) Representative fluorescence images of muscle cells of C. elegans DM8005 worms cultured in the control condition, with PRIOME^® JH or HT-PB01. 20× magnification. Bar scale = 50 µm.

The protective effect of both HT strains was also observed in the assessment of muscular functionality. The mobility of C. elegans cultured with or without PRIOME^® JH and HT-PB01 HT strains was evaluated over the course of one week using the miniTower system. Figure 8 illustrates the mobility of worms under different conditions, expressed as the percentage relative to the mobility of control worms on day 1. Worms supplemented with PRIOME^® JH and HT-PB01 demonstrated higher mobility from the first day, with values reaching 148% and 153%, respectively. Throughout the entire week, the mobility of worms treated with HT strains remained consistently higher than that of the control group. By day 7, the relative mobility of control worms had decreased to 28%, whereas worms treated with PRIOME^® JH and HT-PB01 maintained mobility levels of 46% and 48%, respectively. Moreover, with a mixed-effects analysis, the supplemented conditions resulted in significant factors (p-value < 0.01); although the interaction between the time and conditions was not significant (p-value = 0.186).

Figure 8.

Mobility of C. elegans under the influence of PRIOME^® JH and HT-PB01. Percentage of mobility of C. elegans N2 worms treated with PRIOME^® JH or HT-PB01 with respect to the control condition (standard diet) for 7 days. Data correspond to the average of three independent assays (a total of 600 worms/condition). Statistical analysis: Data were analyzed using a mixed-effects analysis. The level of significance corresponds to p-value < 0.01.

4. Discussion

Canine OA is a chronic degenerative condition of joints involving pain, low-grade inflammation, and progressive joint damage. Current approaches to canine OA primarily focus on mitigating the adverse consequences of pain, mobility impairment, and reduced quality of life. However, veterinary medicine is currently shifting towards a more preventive approach, promoting earlier detection of canine OA and proactive management of risk factors in dogs [17].

Emerging evidence suggests that microorganisms can contribute to joint health, offering new opportunities for innovative strategies in canine OA. Several studies have proposed the use of probiotics as a potential alternative therapy for OA in both animals and humans [25,26,27]. Furthermore, postbiotics also present promising effects in the management of different OA risk factors, including obesity [22,23] and gut inflammation [19,21]. Given that nearly 40% of young dogs already show early signs of OA [34], probiotic or postbiotic supplementation may help preserve joint health in a substantial portion of the canine population.

In the present study, multiple preclinical models tailored to address specific factors associated with canine OA were employed to assess the impact of postbiotics on key mechanisms contributing to the disease progression, including joint inflammation [35,36] and collagen synthesis [5]. Also, systemic factors that influence OA development, such as epithelial gut cells inflammation [6], gut permeability [8], body weight and fat accumulation [37], and muscle degradation [13] were evaluated. Taken together, these preclinical studies established a robust scientific framework for candidate identification and lead selection for the development of future clinical trials.

Among the different preclinical approaches used in this study, C. elegans has emerged as a well-established in vivo model organism. The high degree of conservation of genes and signaling pathways between C. elegans and mammals makes it a suitable system for investigating the molecular mechanisms underlying human disorders [38]. Many genes involved in fat metabolism are conserved between this nematode and mammals [39]. Moreover, nematodes exhibit morphological and functional similarities in the gut, and their muscle architecture is highly conserved [40,41]. Furthermore, recent studies indicated that positive outcomes identified through probiotic and postbiotic evaluation in C. elegans can be translated to cats and dogs [42,43,44]. As a result of this evaluation, the HT strains of L. rhamnosus PRIOME^® JH and PB01 emerged as promising microbiome-based interventions for mitigating risk factors associated with joint health in canines. However, these findings are derived from preclinical models and, therefore, further validation through in vivo studies in dogs would be required to determine their clinical relevance.

First of all, we use a canine-specific cell culture model as a physiological approach to assess the anti-inflammatory capacity of different HT strains. Among the tested HT bacteria, two L. rhamnosus strains, PRIOME^® JH and PB01, exerted a supportive effect, reducing the secretion of the proinflammatory cytokine IL-6. This effect may contribute to a reduction in the proinflammatory status at the joint level, thereby protecting cartilage from inflammation-induced degradation [45]. Moreover, results are in accordance with previous data obtained in a pilot clinical study with PB01, which shows the effects of the probiotic strain L. rhamnosus PB01 in patients with osteoarthritis, leading to a reduction in pain in osteoarthritic knees [25]. Furthermore, the results obtained here in canine chondrocytes suggest that L. rhamnosus PB01 preserves the anti-inflammatory activity in its heat-treated form.

The effect of both HT strains on the immune response was subsequently evaluated in canine macrophage cell cultures. The two potential postbiotics, PRIOME^® JH and HT-PB01, induced a high IL-10/IL-12 ratio, suggesting that canine macrophages challenged with the HT strains were polarized toward an anti-inflammatory M2 phenotype. In this state, macrophages have shown to secrete cartilage-promoting cytokines, such as transforming growth factor (TGF)-β1, TGF-β3, insulin-like growth factor 1 (IGF-1), and IGF-2, shifting the local immune microenvironment toward a pro-chondrogenic condition, thereby facilitating cartilage homeostasis [36]. Furthermore, M2 macrophages may enhance the synthesis of extracellular matrix (ECM) components, such as collagen and proteoglycans, through the secretion of vascular endothelial growth factor (VEGF), TGF-β, and arginine [36].

Building on the anti-inflammatory potential of both HT strains, we aimed to validate their beneficial effects using a biological model for type II collagen production in joints. Type II collagen plays a key structural role in cartilage, and it has been reported to suppress hypertrophic differentiation in healthy chondrocytes [5]. Both L. rhamnosus postbiotics, PRIOME^® JH and HT-PB01, exhibited an enhancing effect on the type II collagen production in canine chondrocytes without affecting MMP-1 secretion levels, indicating the potential beneficial effect of these two HT strains on OA. The accumulation of type II collagen may contribute to the maintenance of chondrocyte homeostasis by regulating signaling pathways related to hypertrophy, such as the SMAD pathways, thereby promoting long-term chondrocyte functionality [5]. Alternatively, the increased type II collagen levels might result from reduced chondrocyte atrophy directly regulated by interaction with inactivated cells. Although an inhibition of MMP-1 enzymatic activity by both postbiotics cannot be ruled out, these findings point out that the underlying mechanism would be an enhancement of type II collagen levels. Future studies should confirm the improvement of ECM functionality by assessing whether the increase in type II collagen is accompanied by a parallel increase in other key ECM components, such as proteoglycans. Proteoglycans, particularly aggrecan, are essential for maintaining cartilage integrity by providing its characteristic compressive properties. Recent studies using probiotic strains have demonstrated preservation of proteoglycan content and attenuation of cartilage degradation through modulation of inflammatory and catabolic pathways, suggesting that probiotic-derived factors may indirectly support proteoglycan homeostasis [46,47,48]. Future studies should investigate whether the postbiotics described in this work also contribute to the functional maintenance of the proteoglycan-rich cartilage matrix.

It is important to acknowledge that OA is a multifactorial condition. Factors such as increased gut permeability, gut inflammation, obesity, and aging can contribute to the disruption of joint homeostasis [8]. To further evaluate the broad beneficial potential of these HT strains, we assessed the impact of PRIOME^® JH and HT-PB01 on several processes associated with OA development, including low-grade inflammation sources and sarcopenia.

The initiation and development of OA have been related to innate immune system activation [7]. Gut inflammation and increased intestinal permeability are closely linked and may contribute to low-grade systemic inflammation. This condition occurs when molecules such as LPS or bacterial endotoxins cross the intestinal barrier and activate macrophages [8,12]. The persistent activation of the gut immune system can elevate the systemic levels of NF-κB, promoting the secretion of catabolic cytokines in the cartilage. Both HT strains, PRIOME^® JH and HT-PB01, showed a beneficial effect, enhancing gut barrier integrity and reducing gut inflammation. These properties may help prevent continuous activation of innate immune cells and attenuate systemic proinflammatory signals. Thus, the reduction in systemic low-grade inflammation could have a protective impact on OA development.

Furthermore, obesity can negatively affect joint health through multiple mechanisms. Although mechanical overloading of joints, such as the knee, is associated with pathological changes, non-weight-bearing joints are also affected in obese individuals, suggesting that obesity contributes to systemic factors involved in OA [9,49]. The most widely accepted hypothesis is that obesity contributes to low-grade systemic inflammation. Activated macrophages within adipose tissues, together with adipocytes themselves, secrete proinflammatory cytokines such as IL-6 and TNF-α, promoting an inflammatory state [49]. In addition to systemic low-grade inflammation, fat depots located near the joints, such as the infrapatellar fat pad, have been shown to exert a potential negative effect on joint inflammation in dogs [10]. The fat-reduction effect promoted by PRIOME^® JH and HT-PB01 may help mitigate obesity-related alterations, preserving joint health.

Finally, the muscle protection effect of HT strains was evaluated. Aging is a natural process that impacts the body’s primary functions, including muscle health and mobility. It is associated with a progressive decline in muscle mass and strength, a process known as sarcopenia. This condition not only impairs physical performance but has also been linked to the progression of OA, as reduced muscle functionality contributes to cartilage degradation through mechanical overloading [13]. In this study, an innovative preclinical C. elegans model was employed to investigate the ability of inactivated cells to prevent age-related alterations in sarcomere structure. The model is based on the evaluation of myofilament organization. Both HT strains PRIOME^® JH and HT-PB01 demonstrated positive effects on preserving the myofilament structure of muscle cells in aged C. elegans, as well as on maintaining muscle functionality. Both postbiotic strains significantly increased worms’ mobility (regulated by muscle cell contraction) during aging. These results are consistent with a previous study from a rat model, in which supplementation with HT-L. plantarum beLP-K was associated with muscle protection and mitigation of dexamethasone-induced sarcopenia [50].

In summary, this study investigated the potential beneficial properties of two HT strains on joint health-related parameters using both in vitro and in vivo preclinical models. Particularly, two L. rhamnosus HT strains, PRIOME^® JH and HT-PB01, exhibited the highest ability to mitigate multiple factors associated with the progression of canine OA, including the reduction in inflammation in chondrocyte and intestinal cell cultures and the promotion of type II collagen synthesis in chondrocytes. Furthermore, additional benefits of both HT strains were demonstrated using the C. elegans model, such as improvement of the intestinal barrier, reduction in fat deposition, and mitigation of age-related decline in muscle structure. Overall, these findings suggest that the tested HT strains may provide potential benefits in maintaining joint health, mobility, and quality of life in dogs. Moreover, the data obtained highlight the potential of inactivated cells as competitive candidates for application in animal nutrition, particularly due to their stability.

Despite these promising results, this study presents certain limitations that should be considered when interpreting the findings. Multiple validated preclinical models, such as C. elegans and the human-derived intestinal cell line HT-29, were employed to address the potential beneficial effects of the postbiotics on risk factors associated with OA. However, further studies are needed in canine OA clinical trials to confirm their efficacy. In addition, the lack of well-established canine intestinal cell lines to evaluate gut inflammation represents a methodological constraint. Although HT-29 cells are widely used in research due to their translational relevance, their tumor-human origin constitutes a limitation. Furthermore, OA often develops under inflammatory conditions, which negatively affects the ECM. In the present study, the characterized postbiotics were able to inhibit IL-6 secretion upon an inflammatory challenge and to stimulate type II collagen under basal conditions. These data suggest that the matrix protein synthesis could be preserved in canine chondrocytes under inflammatory conditions, although further studies are required to support this hypothesis. Finally, from our screening, only two out of nine tested strains demonstrated measurable efficacy under the experimental conditions, which highlights the strong strain-specific nature of postbiotic effects.

Overall, this study identifies PRIOME^® JH and HT-PB01 HT strains as promising postbiotic candidates for further research in dogs. The present evidence provides a basis for future in vivo investigation aimed at evaluating postbiotic strategies to address multiple risk factors associated with canine osteoarthritis.

5. Conclusions

In this study, two potential postbiotics, PRIOME^® JH and HT-PB01, were identified as promising functional ingredients for supporting canine joint health. Both HT strains were able to reduce inflammation in canine chondrocytes and enhance type II collagen production in preclinical models. Additionally, PRIOME^® JH and HT-PB01 modulated canine macrophages, promoting tissue homeostasis, which is closely associated with joint health.

Both HT strains exhibited the capability to reduce gut inflammation, improve intestinal barrier integrity, and decrease fat accumulation, which are well-known OA risk factors associated with systemic low-grade inflammation. Furthermore, PRIOME^® JH and HT-PB01 demonstrated protective effects against sarcopenia in the C. elegans model, showing both structural and functional preservation of muscle tissue.

These findings highlight the potential of PRIOME^® JH and HT-PB01 as functional ingredients for addressing risk factors associated with canine OA. Future clinical trials are necessary to elucidate the clinical relevance of these ingredients in dogs.

Abbreviations

The following abbreviations are used in this manuscript:

ATCC	American Type Culture Collection
BCA	Bicinchoninic Acid
CGC	Caenorhabditis Genetics Center
DMEM	Dulbecco’s Modified Eagle Medium
FBS	Fetal Bovine Serum
HT	Heat-Treated
IGF-1	Insulin-like Growth Factor 1
IL-10	Interleukin 10
IL-12	Interleukin 12
IL-1β	Interleukin-1 beta
IL-6	Interleukin-6
LPS	Lipopolysaccharides
MMPs	Matrix Metalloproteinases
MRS	Man–Rogosa–Sharpe
MTT	3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MTX	Methotrexate
NC	Negative Control
NGM	Nematode Growth Medium
NSAIDs	Non-Steroidal Anti-Inflammatory Drugs
OA	Osteoarthritis
SD	Standard deviation
TGF-β1	Transforming Growth Factor beta 1
TNF-α	Tumor Necrosis Factor Alpha
UPV	Polytechnical University of Valencia
VEGF	Vascular Endothelial Growth Factor

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells15040336/s1, Figure S1: Interleukin-6 secretion levels in the canine chondrocytes model. Figure S2: Canine chondrocyte viability after 18 h incubation with heat-treated strains: PRIOME^® JH and HT-PB01. Figure S3: MMP-1 production by primary canine chondrocytes cultured in spheroids structure. Table S1: Catalog numbers of kits and reagents used in this study.

Author Contributions

Conceptualization, L.R., G.G.-L., M.M., E.C. and P.M.; methodology, L.R., G.G.-L., M.M., E.P., V.N., J.R., F.B., R.M., S.L. and P.M.; validation, L.R., G.G.-L., M.M., A.R., E.C. and P.M.; formal analysis, G.G.-L., M.M. and S.L.; investigation, G.G.-L., M.M., E.P., V.N., J.R., F.B., R.M. and S.L.; resources, L.R., G.G.-L., M.M., E.C. and P.M.; data curation, G.G.-L., M.M., E.P., V.N., J.R., F.B., R.M. and S.L.; writing—original draft preparation, L.R., G.G.-L., M.M., S.L. and A.F.-R.; writing—review and editing, L.R., G.G.-L., M.M., S.L., A.R., A.F.-R., E.C. and P.M.; visualization, L.R., G.G.-L., M.M., S.L., A.R. and A.F.-R.; supervision, E.C. and P.M.; project administration, L.R., G.G.-L., M.M., S.L., E.C. and P.M.; funding acquisition, L.R., E.C. and P.M. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author due to privacy reasons.

Conflicts of Interest

Authors L.R., G.G.-L., M.M., E.P., V.N., J.R., F.B., R.M., S.L., A.R., A.F.-R., E.C and P.M. were employed by the company Archer Daniels Midland (ADM).

Funding Statement

This research was funded by Archer Daniels Midland, Nutrition, R&D Health & Wellness, Biopolis S.L. Parc Cientific Universitat de València.