Orally Administered Porcine Intestinal Lactobacilli Improve the Respiratory Innate Immune Response Against Streptococcus pneumoniae

Simple Summary

Respiratory infections are a major cause of disease in pigs, and new strategies are needed to enhance host resistance in a safe and practical manner and to avoid the overuse of antimicrobials. In this study, we evaluated whether lactobacilli isolated from the porcine intestinal tract could modulate respiratory immunity through the gut–lung axis, in an experimental mice model of pneumococcal infection. Among three Ligilactobacillus salivarius strains tested, only the orally administered strain LAFF998 improved resistance to pneumococcal infection, reducing lung bacterial burden and tissue damage while enhancing the functionality of alveolar macrophages. These findings demonstrate that immunomodulatory effects are strain-specific and identify L. salivarius LAFF998 as a promising immunobiotic candidate to strengthen respiratory innate immunity and prevent bacterial pneumonia in pigs.

Abstract

Background: Respiratory bacterial infections represent a major health challenge in swine production, highlighting the need for novel immunomodulatory strategies that enhance host resistance. In this study, we investigated whether porcine intestinal lactobacilli could modulate the gut–lung axis and improve respiratory innate immunity in a mouse model of Streptococcus pneumoniae infection, as a surrogate of Streptococcus suis pneumonia. Methods: Three strains of Ligilactobacillus salivarius (LAFF998, LAFF1071, and LAFF1095) were orally administered to Swiss mice prior to pneumococcal challenge. The resistance to the infection, the lung damage and the respiratory innate immune response were evaluated. Results: Only strain LAFF998 significantly reduced pulmonary bacterial loads, prevented bacteremia, and attenuated lung injury. This protective effect was associated with selective modulation of respiratory immunity, characterized by reduced neutrophilic inflammation, increased lymphocyte recruitment, and enhanced activation of alveolar macrophages expressing MHC-II. LAFF998 markedly increased the production of IFN-β, IFN-γ, IL-6, IL-10, and IL-27 in the respiratory tract, without inducing excessive inflammatory damage. Ex vivo and in vitro analyses confirmed that alveolar macrophages from LAFF998-treated mice exhibited a primed phenotype with heightened cytokine responses to pneumococcal stimulation. In contrast, strains LAFF1071 and LAFF1095 failed to confer protection or significantly modulate respiratory immune responses. Conclusions: These findings demonstrate a strict strain-dependent effect among porcine L. salivarius isolates and identify LAFF998 as a potent immunobiotic capable of enhancing respiratory innate immunity through the gut–lung axis. This work supports further studies of LAFF998 as an immunobiotic strategy for the prevention of respiratory infections in pigs.

1. Introduction

Streptococcus suis (S. suis) is a Gram-positive bacterium that is one of the most relevant bacterial pathogens in swine production worldwide. Although widely known for causing meningitis, septicemia, and arthritis, S. suis also plays a significant role in respiratory infections, particularly as an agent involved in pneumonia and porcine respiratory disease complex (PRDC) [1,2]. S. suis colonizes piglets’ upper respiratory tract, especially the tonsils and nasopharynx, where it can persist as a commensal or pathobiont. Under stressful conditions, viral coinfections with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) or Swine Influenza Virus (SIV), immunosuppression, or management deficiencies, the bacteria can descend to the lower respiratory tract and contribute to the development of bronchopneumonia. In these cases, S. suis often acts as a secondary pathogen, exacerbating pulmonary inflammation and worsening lesions initially induced by viral agents [3]. S. suis-associated pneumonia is characterized by a mixed inflammatory infiltrate dominated by neutrophils and alveolar macrophages, with thickening/widening of the alveolar septa and, in some cases, suppurative foci consistent with detrimental inflammation [4].

Experimental respiratory infection models of S. suis have been established in pigs using intranasal or intratracheal inoculation, allowing the study of pneumonia, pulmonary inflammation, and host innate immune responses, both in single infections and in viral–bacterial coinfection settings representative of PRDC [5,6]. However, such approaches require the use of large animals, specialized infrastructure, and substantial financial and logistical resources. Therefore, to enable a more practical assessment of streptococcal pneumonia and innate immune regulation, Streptococcus pneumoniae has been employed as a surrogate respiratory pathogen. The bacteria S. suis and S. pneumoniae share several microbiological and pathogenic features, including the induction of neutrophil- and macrophage-dominated pneumonia. Importantly, in both infections, early host protection relies on alveolar macrophage activation, interferon responses and inflammatory cytokines signaling, as well as regulation of excessive inflammation to prevent systemic dissemination [7], supporting the use of pneumococcal infection models as a conceptual framework for studying S. suis respiratory disease. In addition, key biological parallels between the two species exist: both are encapsulated Gram-positive streptococci in which the capsular polysaccharide is a major virulence determinant impacting interactions with host phagocytes, and both elicit lung inflammation dominated by neutrophils and macrophages [8]. Moreover, S. suis expresses suilysin, a cholesterol-dependent cytolysin closely related at the family level to pneumolysin produced by S. pneumoniae [9]. In addition, primary pneumococcal pneumonia and viral–pneumococcal superinfection models are highly standardized in mice and allow reproducible quantification of pulmonary innate immune responses and lung injury [10,11], providing a robust platform to test immunomodulatory interventions prior to confirmatory studies in pigs.

Studies have shown that the intestinal microbiota can influence the outcome of S. suis infection. In mice, it was shown that the alteration of the intestinal microbiota by antibiotic treatment aggravates lung injury during streptococcal respiratory infection [12]. Animals receiving broad-spectrum antibiotics (ampicillin, metronidazole, neomycin, and vancomycin) showed aggravated lung pathology characterized by alveolar thickening, enhanced inflammatory infiltration, and hemorrhage compared to infected controls. Interestingly, these lung histopathological alterations were associated with significantly altered levels of interleukin (IL)-2, IL-12, transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α, and IL-4 in the respiratory tract, highlighting the role of the intestinal microbiota in modulating respiratory immunity. On the other hand, it was also shown that the intestinal microbiota is a critical determinant of respiratory antipneumococcal immunity by priming lung defenses through a granulocyte–macrophage colony-stimulating factor (GM-CSF)—dependent pathway [13]. Using wild-type, antibiotic-treated, and germ-free mice, the authors showed that microbiota depletion markedly impaired S. pneumoniae clearance, whereas oral administration of commensal bacteria with high nucleotide-binding and oligomerization domain (NOD2) receptor-stimulating capacity restored pulmonary immunity. Mechanistically, microbiota-derived signals promoted IL-17A–dependent induction of pulmonary GM-CSF, which in turn enhanced alveolar macrophage-mediated bacterial clearance. In line with these findings, it was shown that the depletion of the intestinal microbiota in C57BL/6 mice resulted in impaired early clearance of S. pneumoniae, increased bacterial dissemination into the bloodstream, exacerbated lung inflammation, and enhanced damage to distant organs during sepsis, leading to reduced survival [14]. Importantly, fecal microbiota transplantation restored pulmonary bacterial clearance as well as TNF-α and IL-10 production in microbiota-depleted mice. Of note, alveolar macrophages from microbiota-depleted animals exhibited marked transcriptional reprogramming, particularly affecting lipid metabolism and innate immune pathways, along with reduced phagocytic capacity and diminished cytokine responses. Collectively, these findings identify intestinal beneficial microbes as a key regulators of respiratory immune mechanisms involved in the protection against streptococci.

We have previously used mice models of S. pneumoniae infection to select and characterize lactobacilli strains with the capacity to modulate the gut–lung axis [15]. Among the strains evaluated, Lacticaseibacillus rhamnosus CRL1505 have demonstrated a remarkable capacity to modulate respiratory immunity. Oral administration of CRL1505 is able to increase the activation of alveolar macrophages and promote the production of key antibacterial mediators, including interferon (IFN)-γ and TNF-α, in the respiratory tract. This effect was associated with improved pathogen clearance and reduced lung damage following respiratory challenge with S. pneumoniae [15]. Of note, the findings in experimental mice models of pneumococcal pneumonia were subsequently validated in human intervention studies, where L. rhamnosus CRL1505 administration was associated with a reduced incidence and severity of respiratory infections [16]. These observations highlight the strong predictive value of murine infection models for identifying immunobiotic strains with translational relevance and clinical efficacy.

The strains LAFF998, LAFF1071 and LAFF1095, belonging to the Ligilactobacillus salivarius species, were previously isolated from the porcine small intestine and preselected for their ability to modulate the innate immune response in porcine intestinal epithelial cells (submitted for publication). In this work, these three strains were studied to evaluate whether they can stimulate the gut–lung axis and improve protection against a respiratory pathogen. For this purpose, a mice model pneumococcal infection was used, and the lung bacterial cell counts and damage as well as the respiratory innate immune response were evaluated.

2. Materials and Methods

2.1. Microorganisms and Culture Conditions

Three Ligilactobacillus salivarius strains (LAFF998, LAFF1071 and LAFF1095) were used in this study. These strains were previously isolated from the small intestine of healthy adult pigs and were selected because of their capacity to modulate immune responses in porcine intestinal epithelial cells in vitro (submitted for publication). These strains belong to the culture collection of the Laboratory of Animal Food Function (LAFF) of Tohoku University (Sendai, Japan).

Each strain was initially inoculated onto MRS agar (Difco, Detroit, MI, USA) and anaerobically incubated at 37 °C. Single colonies were then transferred to MRS broth, and bacteria were grown for 24 h under anaerobic conditions. Following two successive subcultures, the bacteria were cultured for an additional 16 h. Then, lactobacilli cultures were centrifugated at 5800 rpm for 5 min, and the resulting pellets were washed three times with PBS after removal of the supernatant.

Streptococcus pneumoniae serotype 6B was obtained from the National Institute of Infectious Diseases of Argentina (ANLIS-Malbrán, Buenos Aires, Argentina), where the strain was characterized by serotyping and molecular analyses. Pneumococci grew on blood agar plates for 18 h at 37 °C under 5% CO₂. Pneumococcal colonies were subsequently transferred to Todd–Hewitt broth and incubated 16 h at 37 °C with 5% CO₂. Bacterial cells were then collected, washed, and resuspended in PBS to a final concentration of 4 × 10⁷ CFU/mL.

2.2. Experiments in Mice

Swiss-albino mice (21 days old) were obtained from the Argentinean Reference Center for Lactobacilli (CERELA-CONICET, Tucumán, Argentina). Nine mice were assigned to each experimental group, and both treatment and control groups were provided with a balanced diet and water ad libitum. Animals were housed in plastic cages under controlled room-temperature conditions. All experimental procedures were approved by the Ethical Committee of CERELA–CONICET and were conducted in accordance with the Declaration of Helsinki, following the protocols for animal experimentation (CRL-CICUAL-IBT-2024/7A).

Mice received a daily oral administration of live L. salivarius strains LAFF998, LAFF1071, or LAFF1095 (Figure 1). Each mouse received a dose of 1 × 10⁸ cells/mouse/day, for five consecutive days. This dose was selected according to our previous work evaluating immunomodulatory lactobacilli with the capacity to modulate the gut–lung axis in mice [17]. Twenty-four hours after the last administration of lactobacilli, mice were challenged with 1 × 10⁵ pneumococcal cells via the nasal route. Two days after infection, mice were euthanized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg), and lung and blood samples were taken to quantify S. pneumoniae growth (Figure 1). Results were recorded as positive/negative hemocultures or log CFU per gram of lung. In addition, following euthanasia, broncho-alveolar lavage fluid (BAL) was collected by instilling sterile phosphate-buffered saline (PBS) through the respiratory tract to evaluate markers of lung injury, number of leukocytes and the levels of cytokines. Three mice per group per each time point evaluated were used, and three independent experiments were performed (n = 9).

Figure 1.

Experimental design to evaluate the effect of porcine Ligilactobacillus salivarius strains on the resistance against Streptococcus pneumoniae infection. Infant mice (3 weeks old) were orally treated with L. salivarius LAFF998, LAFF1071 or LAFF1095 (1 × 10⁸ cell/mouse/day) during five consecutive days. One day 6, mice were infected with S. pneumoniae serotype 6B. After 48 h of the pneumococcal challenge, the resistance to the infection and the respiratory innate immune response were evaluated.

2.3. Lung Injury Parameters

BAL samples were collected following previously established procedures [18]. Briefly, the trachea was surgically exposed, cannulated, and lavaged twice consecutively with sterile PBS. The recovered lavage fluid was centrifuged at 900× g for 10 min, and the cell-free supernatant was stored at −70 °C until further analysis.

To evaluate lung injury, markers of increased broncho-alveolar–capillary barrier permeability and cytotoxicity were assessed in the cell-free BAL fluid. Total protein and albumin concentrations, indicative of enhanced vascular permeability, as well as lactate dehydrogenase (LDH) activity, a general marker of cellular damage, were quantified. Albumin levels were determined using a colorimetric assay commercial diagnostic kit (Wiener Lab, Buenos Aires, Argentina). Total protein concentration was measured using the BCA Protein Assay (Pierce Biotechnology Inc., Rockford, IL, USA). LDH activity was quantified according to the standards and procedures provided by the Wiener Lab kit (Buenos Aires, Argentina).

2.4. Leukocytes Counts and Cytokine Levels in BAL

The numbers of leukocytes in BAL samples were determined using a blood cell counter and May-Grunwald Giemsa stain was used for the differential cell count, as described previously [18].

BAL samples stored at −70 °C were used for cytokine analysis. The concentrations of BAL IFN-β, IFN-γ, IL-6, IL-10, and IL-27 were measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocols.

2.5. Primary Culture of Alveolar Macrophages

The Primary cultures of murine alveolar macrophages were established following previously described protocols [18]. Briefly, macrophages were obtained from mice by broncho-alveolar lavage using 1 mL of sterile PBS, washed twice, and resuspended in RPMI 1640 medium containing FBS (10%), L-glutamine (1 mM), and penicillin–streptomycin (100 U/mL).

The cells were seeded at a density of 1 × 10⁵ cells per well in 24-well culture plates and incubated for 2 h at 37 °C in a humidified atmosphere containing 5% CO₂ to allow macrophage adhesion. Non-adherent cells were then removed, and alveolar macrophages were subsequently challenged with S. pneumoniae at an MOI = 5, according to our previous studies [15]. Total RNA was extracted 12 h after stimulation, and the expression levels of cytokines and antiviral-related genes were quantified by RT-qPCR.

2.6. RT–qPCR

Gene expression of cytokines and chemokines, including IFN-β, IFN-γ, IL-6, IL-10, IL-12, IL-27, TNF-α, IL-1β, CCL2, CCL3, CXCL2, and CXCL10, was analyzed in primary alveolar macrophage cultures using two-step quantitative real-time PCR. Total RNA was extracted with TRIzol reagent (Invitrogen, Waltham, MA, USA), and cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen, Tokyo, Japan). RT–qPCR was performed on a 7300 Real-Time PCR System (Applied Biosystems, Warrington, UK) using Platinum SYBR Green qPCR SuperMix with uracil-DNA glycosylase and ROX (Invitrogen). Primer sequences were described previously [18]. Cycling conditions consisted of 2 min at 50 °C, 2 min at 95 °C, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Gene expression levels were normalized to β-actin.

2.7. Statistical Analysis

The experiments were performed in triplicate, and the results are expressed as the mean ± SD (SD). Data distribution normality was assessed prior to applying two-way ANOVA, and homogeneity of variance was verified to ensure the validity of parametric analyses. Tukey’s test was used to test for differences between groups. Differences were judged as significant at p < 0.05.

3. Results

3.1. Porcine L. salivarius LAFF998 Increases the Resistance to Pneumococcal Infection

L. salivarius LAFF998, LAFF1071 and LAFF1095 were orally administered to mice for 5 consecutive days, and on day 6 animals were then challenged by the nasal route with S. pneumoniae. Two days after the pneumococcal infection, the lung bacterial cell counts and hemocultures were evaluated.

In control mice, pneumococci was detected in the lungs in levels of approximately 6 log CFU/gram of organ (Figure 2) as well as in blood since hemocultures were positive for this pathogen. The pneumococcal infection also enhanced the levels of the biochemical markers of lung injury lactate dehydrogenase (LDH) and albumin measured in broncho-alveolar lavage (BAL) samples (Figure 2). Of note, when mice received the oral administration of L. salivarius LAFF998 before the pneumococcal challenge, the lung bacterial cell counts, and the levels of BAL LDH and albumin were significantly lower than controls (Figure 2). In addition, mice in the LAFF998 group had negative hemocultures. On the other hand, the oral treatment with L. salivarius LAFF1071 or LAFF1095 did not modify the resistance of animals to S. pneumoniae infection since all the parameters evaluated were not different from controls.

Figure 2.

Effect of porcine Ligilactobacillus salivarius strains on the resistance against Streptococcus pneumoniae infection. Infant mice (3 weeks old) were orally treated with L. salivarius LAFF998, LAFF1071 or LAFF1095 and then infected with S. pneumoniae serotype 6B. After 48 h of the pneumococcal challenge, bacterial cell counts were determined in lung samples. In addition, the levels of lactate dehydrogenase (LDH) and albumin were evaluated in broncho-alveolar fluid lavage (BAL) samples. Data represent three independent experiments (n = 9 per group). Significant differences compared to the control group, ** p < 0.01.

3.2. Porcine L. salivarius LAFF998 Modulates Innate Immunity Against Pneumococcal Infection

We next aimed to evaluate whether the effect of orally administered L. salivarius LAFF998 on the resistance to pneumococcal infection was related to the modulation of respiratory immunity. For this purpose, we first examined changes in the number of BAL leukocytes (Figure 3). As described in our previous works [15], S. pneumoniae infection increased the numbers of BAL leukocytes, neutrophils, macrophages and lymphocytes indicating the activation of the respiratory innate immune response. The oral treatment with L. salivarius LAFF1071 or LAFF1095 did not modify the numbers of BAL leukocytes compared to the control animals except for the increment of BAL lymphocytes induced by the LAFF1071 strain. In addition, mice in the LAFF998-treated group showed values of BAL leukocytes and neutrophils that were lower than the controls while BAL lymphocytes were significantly higher (Figure 3).

Figure 3.

Effect of porcine Ligilactobacillus salivarius strains on respiratory leukocytes numbers after the challenge with Streptococcus pneumoniae. Infant mice (3 weeks old) were orally treated with L. salivarius LAFF998, LAFF1071 or LAFF1095 and then infected with S. pneumoniae serotype 6B. After 48 h of the pneumococcal challenge, the levels of leukocytes, macrophages, neutrophils and lymphocytes were evaluated in broncho-alveolar fluid lavage (BAL) samples. Data represent three independent experiments (n = 9 per group). Significant differences compared to the control group, * p < 0.05.

Specific changes in BAL alveolar macrophages were further evaluated by flow cytometry (Figure 4). No differences between the control and lactobacilli-treated groups were found when the total population of CD45⁺CD11c⁺SiglecF⁺ macrophages were evaluated in BAL samples. Interestingly, when mice received the oral administration of L. salivarius LAFF998 before the pneumococcal challenge the numbers of BAL CD11c⁺SiglecF⁺ macrophages expressing MHC-II were significantly higher than the control group (Figure 4). On the other hand, the oral treatment with L. salivarius LAFF1071 or LAFF1095 did not modify the numbers of CD11c⁺SiglecF⁺MHC-II⁺ macrophages compared to controls (Figure 4).

Figure 4.

Effect of porcine Ligilactobacillus salivarius strains on alveolar macrophages (AMs) numbers and activation after the challenge with Streptococcus pneumoniae. Infant mice (3 weeks old) were orally treated with L. salivarius LAFF998, LAFF1071 or LAFF1095 and then infected with S. pneumoniae serotype 6B. After 48 h of the pneumococcal challenge, the levels of CD45⁺CD11c⁺SiglecF⁺ and CD11c⁺SiglecF⁺MHC-II⁺ AMs were evaluated in broncho-alveolar fluid lavage (BAL) samples. Data represent three independent experiments (n = 9 per group). Significant differences compared to the control group, * p < 0.05.

We also studied the effect of orally administered lactobacilli in the production of inflammatory and regulatory cytokines in the respiratory tract in response to the pneumococcal infection (Figure 5). When mice received the oral administration of L. salivarius LAFF998 prior to the pneumococcal challenge, the expression levels of IFN-β, IFN-γ and IL-6 were significantly higher than those observed in the control animals, while the levels of IL-12 were not different from the controls (Figure 5). In addition, the LAFF998 strain was able to induce increases in the regulatory cytokines IL-10 and IL-27. In contrast, mice receiving LAFF1071 or LAFF1095 strains displayed respiratory cytokine profiles similar to those of control animals (Figure 5), highlighting the strain specificity of the observed effects.

Figure 5.

Effect of porcine Ligilactobacillus salivarius strains on respiratory cytokine profiles after the challenge with Streptococcus pneumoniae. Infant mice (3 weeks old) were orally treated with L. salivarius LAFF998, LAFF1071 or LAFF1095 and then infected with S. pneumoniae serotype 6B. After 48 h of the pneumococcal challenge, the levels of interferon (IFN)-β, IFN-γ, interleukin (IL)-6, IL-10, IL-12 and IL-27 were evaluated in broncho-alveolar fluid lavage (BAL) samples. Data represent three independent experiments (n = 9 per group). Significant differences compared to the control group, * p < 0.05, ** p < 0.01.

3.3. Porcine L. salivarius LAFF998 Modulates the Response of Alveolar Macrophages to Pneumococcal Infection

Considering that the oral administration of L. salivarius LAFF998 seemed to exert an effect particularly on alveolar macrophages, we next aimed to investigate whether the changes in the respiratory cytokine profile was associated with changes in alveolar macrophages function. Then, mice were orally treated with lactobacilli, alveolar macrophages were collected, cultured and in vitro stimulated with S. pneumoniae. The response of alveolar macrophages to the pneumococcal challenge was assessed by measuring cytokine levels in culture supernatants (Figure 6). When the basal production of cytokines was measured before the challenge with S. pneumoniae, it was noticed that macrophages obtained from LAFF998-treated mice produced higher levels of IFN-β, IFN-γ, IL-6, IL-10 and IL-27, but not IL-12 (Figure 6). The levels of cytokines produced by alveolar macrophages obtained from animals orally treated with L. salivarius LAFF1071 or LAFF1095 in basal conditions were not different from controls.

Figure 6.

Effect of porcine Ligilactobacillus salivarius strains on cytokine profiles of alveolar macrophages (AMs) after the challenge with Streptococcus pneumoniae. Infant mice (3 weeks old) were orally treated with L. salivarius LAFF998, LAFF1071 or LAFF1095. AMs were isolated and infected in vitro with S. pneumoniae serotype 6B. After 24 h of the pneumococcal challenge, the levels of interferon (IFN)-β, IFN-γ, interleukin (IL)-6, IL-10, IL-12 and IL-27 were evaluated on the supernatants of AMs cultures. In addition, basal cytokine production (without pneumococcal challenge) in AMs obtained from LAFF998-treated and control mice are shown. Data represent three independent experiments (n = 9 per group). Significant differences between the indicated groups, * p < 0.05.

The in vitro challenge with pneumococci increased the levels of all the cytokines evaluated compared with basal levels in all the experimental groups. However, alveolar macrophages from LAFF998-treated mice produced higher levels of IFN-β, IFN-γ, IL-6, IL-10 and IL-27 than controls (Figure 6).

Changes in cytokine and chemokine production in alveolar macrophages were further evaluated by qPCR (Figure 7). In line with the evaluation of protein synthesis, the levels of mRNA of IFN-β, IFN-γ, IL-6, IL-10 and IL-27 in alveolar macrophages form L. salivarius LAFF998-treated mice were significantly higher compared with the control group, whereas no differences were observed in IL-12 levels (Figure 7). Regarding pro-inflammatory mediators, alveolar macrophages from LAFF998-treated animals expressed higher levels of TNF-α, IL-1β, CCL2 and CXCL2 compared with control mice, whereas no significant differences were detected in CCL3 or CXCL10 levels (Figure 7). On the other hand, alveolar macrophages obtained after the oral treatment with L. salivarius LAFF1071 or LAFF1095 did not significantly modify the levels of these cytokines compared with controls (Figure 7).

Figure 7.

Effect of porcine Ligilactobacillus salivarius strains on the transcriptomic response of alveolar macrophages (AMs) after the challenge Streptococcus pneumoniae. Infant mice (3 weeks old) were orally treated with L. salivarius LAFF998, LAFF1071 or LAFF1095. AMs were isolated and infected in vitro with S. pneumoniae serotype 6B. After 12 h of the pneumococcal challenge, the expression levels of cytokines and chemokines were evaluated by qPCR. Data represent three independent experiments (n = 9 per group). Significant differences between the indicated groups, * p < 0.05, ** p < 0.01.

4. Discussion

Porcine respiratory infection models for S. suis have been established, including intranasal challenge in swine and coinfection settings representative of PRDC; however, these experiments require the handling of pigs and specialized facilities and are costly and logistically demanding. As a tractable alternative to evaluate streptococcal pneumonia and innate immune regulation, we used S. pneumoniae as a surrogate respiratory pathogen. Then, we used a mice model of pneumococcal respiratory infection to evaluate the capacity of porcine lactobacilli to modulate immunity in the respiratory tract through the gut–lung axis. Of the three L. salivarius strains tested, only LAFF998 significantly improved resistance to pneumococcal infection by lowering lung bacterial loads, limiting systemic spread, and reducing pulmonary tissue injury. The lack of protective effects observed with LAFF1071 and LAFF1095 further highlights the strict strain specificity of immunobiotic activity. Although the mechanisms underlying these differences were not investigated in the present study, it is plausible that variations in cell-surface structures, secreted metabolites, or pattern-recognition receptor-stimulating capacities may account for the differential ability to activate gut–lung immune pathways. Future “omics” studies, including comparative genomics or secretomics, could pinpoint the exact cell-surface molecules responsible for LAFF998’s superior performance. In addition, functional analyses will be necessary to elucidate the molecular determinants responsible for the superior immunomodulatory profile of LAFF998.

The protection induced by L. salivarius LAFF998 against pneumococcal respiratory infection was linked to the modulation of respiratory innate immunity, in particular to the enhanced activation of alveolar macrophages. This is in line with previous studies demonstrating the role of the intestinal microbiota in shaping the effector functions of alveolar macrophages in front of S. pneumoniae infection [13,14]. Furthermore, the results agree with our own previous studies showing that orally administered immunomodulatory lactobacilli such as L. rhamnosus CRL1505 [15] modulate the cytokine profile in alveolar macrophages upon the challenge with pneumococci.

It was observed that alveolar macrophages from LAFF998-treated mice produced higher levels of IFN-β, IFN-γ, IL-6, TNF-α, IL-1β, CCL2 and CXCL2, all of which have been associated with the protection against pneumococcal pneumonia. Early studies with IFN-γ^−/− C57BL/6 mice reported a higher susceptibility to S. pneumoniae infection in animals lacking IFN-γ than in wild-type mice [19]. Similarly, TNF-α⁻/⁻ mice displayed increased susceptibility to pneumococcal infection, with reduced survival compared to wild-type controls. The absence of TNF-α resulted in higher pulmonary bacterial loads and enhanced systemic dissemination that led to exacerbated inflammation, and more severe tissue injury [20]. Furthermore, it was demonstrated that the enhanced production of IFN-γ and TNF-α by alveolar macrophages resulted in lower pneumococcal burdens in the infected mice and significantly improved survival rates [21]. On the other hand, the cytosolic DNA-sensing pathway during S. pneumoniae infection is responsible for the induction of the production of type I IFNs (IFN-β) in the respiratory tract. It was shown that both human and murine macrophages produced IFN-β in response to infection, in a process dependent on bacterial phagocytosis, endosomal acidification, and activation of the STING–IRF3 signaling axis [22]. IFN-β produced in response to pneumococcal infection has several protective effects including the reduction inflammation-induced death of alveolar epithelial type II cells [23] and the limitation of bacterial dissemination from the lung into the bloodstream [24]. In fact, mice lacking the type I IFN receptor (Ifnar1⁻/⁻) or treated with receptor-neutralizing antibodies developed enhanced bacteremia following intranasal infection, despite similar pulmonary bacterial loads compared with wild-type animals. IL-1β and IL-6 also play pivotal roles in the host response to S. pneumoniae. IL-1 β participates in the coordinated interaction between alveolar macrophages and pulmonary epithelial cells, in which macrophage-derived IL-1β drives epithelial chemokine release, promoting neutrophil recruitment [25]. IL-6 deficiency was shown to increase macrophage death and exacerbate lung inflammation, whereas exogenous IL-6 administration reduced macrophage loss and attenuated tissue injury [26]. Collectively, these findings indicate that LAFF998 would promote a coordinated and protective cytokine milieu in alveolar macrophages that are essential for bacterial clearance, preservation of epithelial integrity, and prevention of systemic dissemination during pneumococcal pneumonia. Of note, inflammatory cytokine levels were measured at a single post-infection time point. Although the increased mediators could be associated with improved bacterial control and reduced lung injury, several of these cytokines can be protective only within a regulated range; therefore, kinetic studies are needed to confirm that the primed macrophage state induced by LAFF998 does not lead to excessive inflammation at later stages of infection.

Interestingly, beyond enhancing the secretion of pro-inflammatory mediators by alveolar macrophages, L. salivarius LAFF998 also promoted a marked increase in the regulatory cytokines IL-10 and IL-27 within the airways. Moreover, alveolar macrophages isolated from LAFF998-treated mice exhibited elevated production of both IL-10 and IL-27, cytokines recognized for their roles in limiting immune-mediated pulmonary injury [27]. The preservation of tissue integrity during infection requires a tightly controlled equilibrium between inflammatory and regulatory signals, ensuring effective pathogen clearance while preventing excessive collateral damage. In the context of pneumococcal pneumonia, IL-10 has been shown to restrain exaggerated inflammatory responses and enhance survival outcomes [28,29], whereas IL-27, particularly in cooperation with IL-6, contributes to the induction of regulatory T cell responses orchestrated by alveolar macrophages [30]. Then, the functional analyses performed here showed that macrophages from LAFF998-treated animals adopted a primed state, responding more effectively to pneumococcal challenge without promoting excessive inflammation.

This study has certain limitations that should be acknowledged. The experiments in the pneumococcal infection mice model strongly demonstrates the ability of L. salivarius LAFF998 in shaping immune outcomes through the modulation of the gut–lung axis; however, the mechanisms involved in this beneficial effect were not assessed. Considering the similarities of L. salivarius LAFF998 with the previously studied strain L. rhamnosus CRL1505 in shaping respiratory immunity through the modulation of alveolar macrophages function, it is tempting to speculate that the LAFF998 strain would be capable of promoting the mobilization of activated CD4+ cells from the intestinal mucosa to the lungs, where they functionally reprogram alveolar macrophages through the local production of IFN-γ [31]. Nevertheless, further studies are required to determine whether modulation of the intestinal microbiota also contributes to these effects and to clarify the relative importance of microbiota-dependent and immune cell–mediated mechanisms in the gut–lung axis induced by LAFF998. On the other hand, three-week-old mice were selected because this age corresponds to the post-weaning stage, a critical developmental window characterized by immune maturation and increased susceptibility to respiratory infections. This experimental setting was chosen to conceptually model the post-weaning period in piglets, during which modulation of the gut–lung axis may be particularly beneficial for shaping early-life respiratory immunity, although we acknowledge that age-related immune immaturity could influence the magnitude of the observed responses. Thus, it is necessary to investigate whether the oral administration of L. salivarius LAFF998 to pigs of different ages can stimulate the gut–lung axis, improve alveolar macrophage’s function and increase the resistance against S. suis, which is the final goal of our investigations. In this regard, murine alveolar macrophages differ from porcine counterparts in phenotype, receptor expression, and functional responses. Therefore, validation studies in pigs will be necessary to confirm whether the immunomodulatory effects of LAFF998 on alveolar macrophages are conserved across species. Future studies addressing these aspects could position L. salivarius LAFF998 as a probiotic for enhancing respiratory health during swine production.

5. Conclusions

In conclusion, this study demonstrates that porcine intestinal lactobacilli can differentially modulate respiratory innate immunity through the gut–lung axis in a strain-dependent manner. Among the three L. salivarius strains evaluated, only LAFF998 significantly enhanced host resistance to pneumococcal infection, reducing pulmonary bacterial burden, preventing systemic dissemination, and attenuating lung tissue damage. The protective effect of LAFF998 was associated with a selective reprogramming of respiratory innate immunity, characterized by balanced leukocyte recruitment, increased activation of alveolar macrophages, and a coordinated cytokine response. Ex vivo and in vitro analyses further revealed that alveolar macrophages from LAFF998-treated animals acquired a primed functional state, exhibiting heightened responsiveness to pneumococcal stimulation without exacerbating inflammatory damage.

These findings highlight the strict strain specificity underlying immunobiotic effects and reinforce the value of functional screening approaches beyond taxonomic classification. Importantly, the use of a well-established murine pneumococcal pneumonia model provides a robust and predictive platform for the identification of porcine-derived immunobiotics with translational relevance. Collectively, our results identify L. salivarius LAFF998 as a promising immunobiotic candidate for strengthening respiratory innate immunity and preventing bacterial pneumonia, supporting its further evaluation as a practical and safe strategy to improve respiratory health in swine production systems.

Author Contributions

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

Institutional Review Board Statement

All experimental procedures were approved by the Ethical Committee of CERELA–CONICET (Tucuman, Argentina) and were conducted in accordance with the Declaration of Helsinki, following the protocols for animal experimentation. The protocol number approval for the experiments in this work is as follow: CRL-CICUAL-IBT-2024/7A, approved in September 2024.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This study was supported by the ANPCyT–FONCyT grants PICT-2016-0410 and PICT 2021-I-A-00705 to J.V. This study was also supported by a Grant-in-Aid for Scientific Research (A) (23H00354), and a Challenging Research (Exploratory, 23K18072, 25K22412) to H.K. from the Japan Society for the Promotion of Science (JSPS); by the Research Program on Development of Innovative Technology grants (JPJ007097) from the Project of the Bio-oriented Technology Research Advancement Institution (BRAIN), and by the Japan Racing Association to H.K.; and by AMED Grant Number JP21zf0127001.