Orally Administered Lactobacilli Strains Modulate Alveolar Macrophages and Improve Protection Against Respiratory Superinfection

Abstract

Orally administered immunomodulatory lactobacilli can stimulate respiratory immunity and enhance the resistance to primary infections with bacterial and viral pathogens. However, the potential beneficial effects of immunomodulatory lactobacilli against respiratory superinfection have not been evaluated. In this work, we showed that the feeding of infant mice with Lacticaseibacillus rhamnosus CRL1505 or Lactiplantibacillus plantarum MPL16 strains can reduce susceptibility to the secondary pneumococcal infection produced after the activation of TLR3 in the respiratory tract or after infection with RVS. The treatment of mice with CRL1505 or MPL16 strains by the oral route improved the production of interferons in the respiratory tract, differentially modulated the balance of pro- and anti-inflammatory cytokines, reduced bacterial replication, and diminished lung damage. Additionally, we demonstrated that orally administered lactobacilli confer longstanding protection against secondary Streptococcus pneumoniae infection and that this effect would be mediated by the stimulation of trained alveolar macrophages. This work contributes to revealing the mechanisms involved in the modulation of the gut-lung axis by beneficial microbes by demonstrating that specific lactobacilli strains, through the stimulation of the common mucosal immune system, would be able to support the development of trained alveolar macrophages that would confer longstanding protection against secondary bacterial challenges produced after a primary inflammatory event in the respiratory mucosa.

Keywords: Lacticaseibacillus rhamnosus CRL1505; alveolar macrophages; probiotic lactobacilli; respiratory immunity; superinfection.

Figure 1

Effect of Lacticaseibacillus rhamnosus CRL1505 and Lactiplantibacillus plantarum CRL1506 on respiratory superinfection. Infant mice were fed L. rhamnosus CRL1505 or L. plantarum CRL1506 for 5 days and stimulated with poly(I:C) on days 7, 8, and 9 (A) or challenged with respiratory syncytial virus (RSV) on day 7 (B) via the nasal route. Five days later, mice were nasally infected with Streptococcus pneumoniae. The pneumococcal cell counts in lung and blood, the concentration of BAL albumin, and the activity of BAL LDH were determined 2 days after S. pneumoniae infection. The results are shown as mean ± SD. Significant differences are shown compared to the control group at p < 0.05 (*) or p < 0.01 (**).

Figure 2

Effect of lactobacilli on respiratory superinfection. Infant mice were fed L. rhamnosus CRL1505, IBL027, CRL489, L. plantarum CRL1506, or MPL16 for 5 days and stimulated with poly(I:C) on days 7, 8, and 9 via the nasal route. Five days later, mice were nasally infected with Streptococcus pneumoniae. The pneumococcal cell counts in lung and blood, the concentration of BAL albumin, the activity of BAL LDH (A), and the concentrations of BAL IFN-β, IFN-γ, and IL-10 (B) were determined 2 days after S. pneumoniae infection. The results are shown as mean ± SD. Significant differences are shown compared to the control group at p < 0.05 (*) or p < 0.01 (**).

Figure 3

Effect of lactobacilli on AMphs cytokine production. Infant mice were fed L. rhamnosus CRL1505, IBL027, CRL489, L. plantarum CRL1506, or MPL16 for 5 days and stimulated with poly(I:C) on days 7, 8, and 9 via the nasal route. Five days later, AMphs were isolated from BAL samples, cultured, and in vitro challenged with Streptococcus pneumoniae. The concentrations of IFN-β, IFN-γ, IL-6, IL-10, IL-12, and IL-27 were evaluated on AMph supernatants after 24 h. (A) Cytokine production of AMphs from L. rhamnosus CRL1505 and L. plantarum MPL16. The results are shown as mean ± SD. Significant differences were shown compared to the respective basal levels without pneumococcal challenge at p < 0.05 (†). Significant differences were shown compared to the control group at p < 0.05 (*). (B) Heatmap shows the variations in the concentration of cytokines of all experimental groups in relation to the control.

Figure 4

Effect of lactobacilli on AMphs MHC-II expression. Infant mice were fed L. rhamnosus CRL1505, IBL027, CRL489, L. plantarum CRL1506, or MPL16 for 5 days and stimulated with poly(I:C) on days 7, 8, and 9 via the nasal route. Five days later, mice were nasally infected with Streptococcus pneumoniae. The numbers of CD45⁺CD11c⁺SiglecF⁺ and CD11c⁺SiglecF⁺MHC-II⁺ cells in BAL were determined on the last day of lactobacilli treatment (basal) and 2 days after poly(I:C) stimulation and S. pneumoniae infection. The results are shown as mean ± SD. Significant differences are shown compared to the control group at p < 0.05 (*).

Figure 5

Effect of lactobacilli on respiratory superinfection. Infant mice were fed L. rhamnosus CRL1505, IBL027, CRL489, L. plantarum CRL1506, or MPL16 for 5 days and stimulated with poly(I:C) on days 7, 8, and 9 via the nasal route. For the evaluation of long-term protection, 5, 10, 15, or 20 days after the last administration of poly(I:C), mice were nasally infected with Streptococcus pneumoniae. The pneumococcal cell counts in lung and blood, the concentration of BAL albumin, the activity of BAL LDH (A), and the concentrations of BAL IFN-β, IFN-γ and IL-10 (B) were determined 2 days after S. pneumoniae infection. The results are shown as mean ± SD. Significant differences are shown compared to the control group at p < 0.05 (*) or p < 0.01 (**). (C) Heatmap shows the variations in the parameters evaluated for all experimental groups in relation to the controls.

Figure 6

Effect of lactobacilli on respiratory superinfection. Infant mice were fed L. rhamnosus CRL1505, IBL027, CRL489, L. plantarum CRL1506, or MPL16 for 5 days and stimulated with poly(I:C) on days 7, 8, and 9 via the nasal route. Twenty days after the last administration of poly(I:C), mice were nasally infected with Streptococcus pneumoniae. (A) The number of macrophages and neutrophils and the concentrations of TNF-α and CCL2 in BAL samples were determined 3, 6, 12, 24, 32, 48, and 54 h after S. pneumoniae infection. The results are shown as mean ± SD. Significant differences are shown compared to the control group at p < 0.05 (*) (B) Heatmap shows the variations in the parameters evaluated at hours 24 and 48 of all experimental groups in relation to the control.