Lactobacillus rhamnosus D3189 modulates antiviral and inflammatory responses in primary nasal epithelial cells, reducing respiratory syncytial virus shedding

Abstract

Introduction: Respiratory syncytial virus (RSV) infection in the upper respiratory tract promotes disease progression and transmission, with excessive inflammation contributing to severe lower respiratory tract involvement. This study investigates the immunomodulatory effects of Lactobacillus rhamnosus D3189 on viral kinetics and innate immune responses in well-differentiated nasal epithelial cells (WD-NECs).

Methods: WD-NECs from healthy adult donors (N = 8) were cultured in vitro, treated with L. rhamnosus D3189, and then infected with RSV (strain RS4) 24 hours later. Viral replication and shedding were assessed via RT-qPCR and plaque assays. Cytotoxicity and epithelial integrity were evaluated using LDH release and transepithelial electrical resistance (TEER). Inflammatory and antiviral responses were investigated using multiplex immunoassays, AlphaLISA, and ELISA.

Results: RSV infection induced robust viral replication and shedding, disrupted epithelial barrier integrity, and triggered the release of pro-inflammatory cytokines and type I/III interferons. L. rhamnosus D3189 alone did not induce cytotoxicity or inflammation. While it had no effect on viral replication, TEER, LDH release, or IFN-λ1/3 levels, D3189 significantly enhanced IFN-β production, reduced viral shedding, and attenuated RSV-induced cytokine and chemokine responses.

Discussion: L. rhamnosus D3189 modulates the epithelial immune response to RSV, reducing inflammation and viral shedding without compromising epithelial integrity. These findings support its potential as a novel strategy to limit RSV-associated infection and transmission.

Keywords: antiviral; inflammation; innate immunity; lactobacilli; nasal epithelium; respiratory syncytial virus.

Figure 1

Effect of L. rhamnosus D3189 on RSV load 3 days post-infection. WD-NECs were pretreated with D3189 (80 µL of 2.5 × 10⁷ CFU mL^-1) or media (negative control) for 24 h before RSV infection (80 µL of 7.5 × 10⁶ PFU mL^-1) or mock exposure (virus-negative control). At 3 days post-infection, apical washes were collected. (A) Shed virus was quantified by immuno-plaque assay. (B) Viral transcription was assessed by RT-qPCR using RSV N-specific primers, normalised to β-actin expression. Data are presented as median and interquartile range, analysed by Wilcoxon signed-rank test. Each data point represents the mean of duplicate cultures per donor, with donors distinguished by colour (n = 8). **, p < 0.01.

Figure 2

Effect of L. rhamnosus D3189 on LDH release and TEER 3 days post-infection with RSV. WD-NECs were pretreated with D3189 (80 µL of 2.5 × 10⁷ CFU mL^-1) or media (negative control) for 24 h before RSV infection (80 µL of 7.5 × 10⁶ PFU mL^-1) or mock exposure (virus-negative control). At 3 days post-infection, (A) LDH release in the basal media was quantified by LDH-Glo Cytotoxicity Assay Kit, and (B) TEER was measured using the EVOM² Epithelial Voltohmmeter and is presented as a percentage fold change relative to baseline TEER. Data are presented as median and interquartile range, analysed by the Friedman test and uncorrected Dunn’s post hoc test. Each data point represents the mean of duplicate cultures per donor, with donors distinguished by colour (n = 8). *, p < 0.05; **, p < 0.01; ***, p < 0.005.

Figure 3

Effect of L. rhamnosus D3189 IFN-β and IFN-λ1/3–3 days post-infection with RSV. WD-NECs were pretreated with D3189 (80 µL of 2.5 × 10⁷ CFU mL^-1) or media (negative control) for 24 h before RSV infection (80 µL of 7.5 × 10⁶ PFU mL^-1) or mock exposure (virus-negative control). At 3 days post-infection, secreted (A) IFN-β and (B) IFN-λ1/3 were quantified by AlphaLISA and standard ELISA, respectively, using both apical wash and basal media. Data are presented as median and interquartile range, analysed by the Friedman test and uncorrected Dunn’s post hoc test. Each data point represents the mean of duplicate cultures per donor, with donors distinguished by colour (n = 8). *, p < 0.05; **, p < 0.01; ***, p < 0.005.

Figure 4

Effect of L. rhamnosus D3189 pro-inflammatory mediator release 3 days post-infection with RSV. WD-NECs were pretreated with D3189 (80 µL of 2.5 × 10⁷ CFU mL^-1) or media (negative control) for 24 h before RSV infection (80 µL of 7.5 × 10⁶ PFU mL^-1) or mock exposure (virus-negative control). At 3 days post-infection, secreted cytokines and chemokines were quantified in basal media using the Bio-plex pro human cytokine 17-plex assay. Data points represent secreted (A) IFN-γ, (B) IL-1β, (C) IL-4, (D) IL-6, (E) IL-17, (F) MCP-1, (G) MIP-1β and (H) TNF-α from each donor. A (I) heatmap illustrates fold changes in cytokine and chemokine production relative to uninfected controls for all analytes detected with the assay’s limit of detection. Data are presented as median and interquartile range, analysed by the Friedman test and uncorrected Dunn’s post hoc test. Each data point represents the mean of duplicate cultures per donor, with donors distinguished by colour (n = 8). *, p < 0.05; **, p < 0.01; ***, p < 0.005.