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. 2019 Oct 11;15(10):e1008072.
doi: 10.1371/journal.ppat.1008072. eCollection 2019 Oct.

Lung transcriptional unresponsiveness and loss of early influenza virus control in infected neonates is prevented by intranasal Lactobacillus rhamnosus GG

Ogan K Kumova  1 , Adam J Fike  1 , Jillian L Thayer  1 , Linda T Nguyen  2 , Joshua Chang Mell  1 , Judy Pascasio  3 , Christopher Stairiker  1   4 , Leticia G Leon  4 , Peter D Katsikis  4 , Alison J Carey  1   2
Affiliations

Lung transcriptional unresponsiveness and loss of early influenza virus control in infected neonates is prevented by intranasal Lactobacillus rhamnosus GG

Ogan K Kumova et al. PLoS Pathog. .

Abstract

Respiratory viral infections contribute substantially to global infant losses and disproportionately affect preterm neonates. Using our previously established neonatal murine model of influenza infection, we demonstrate that three-day old mice are exceptionally sensitive to influenza virus infection and exhibit high mortality and viral load. Intranasal pre- and post-treatment of neonatal mice with Lactobacillus rhamnosus GG (LGG), an immune modulator in respiratory viral infection of adult mice and human preterm neonates, considerably improves neonatal mice survival after influenza virus infection. We determine that both live and heat-killed intranasal LGG are equally efficacious in protection of neonates. Early in influenza infection, neonatal transcriptional responses in the lung are delayed compared to adults. These responses increase by 24 hours post-infection, demonstrating a delay in the kinetics of the neonatal anti-viral response. LGG pretreatment improves immune gene transcriptional responses during early infection and specifically upregulates type I IFN pathways. This is critical for protection, as neonatal mice intranasally pre-treated with IFNβ before influenza virus infection are also protected. Using transgenic mice, we demonstrate that the protective effect of LGG is mediated through a MyD88-dependent mechanism, specifically via TLR4. LGG can improve both early control of virus and transcriptional responsiveness and could serve as a simple and safe intervention to protect neonates.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Decreased survival of influenza virus-infected neonatal mice coupled with loss of early viral control.
3-day old neonatal and 8 week-old adult mice were intranasally infected with PR8 influenza virus. (A) Neonates were infected with the same sublethal adult virus dose. Neonatal mice (n = 8) and adult mice (n = 4) from 2 independent experiments. (B) Neonates were infected with one fifth of a sublethal adult virus dose. Neonatal mice (n = 24) and adult mice (n = 6) from 3 independent experiments. (C) 3-day old neonatal and 8 week-old adults mice were intranasally infected with the same dose of influenza virus as (B). Mice were harvested at 1, 3, and 6 days post-infection shown. Viral loads in lungs were measured by RT-PCR.
Fig 2
Fig 2. Improved survival and decreased viral loads of neonatal mice when pretreated with Lactobacillus prior to Influenza infection.
(A) Mouse pups received 1 x106 Colony Forming Units of LGG intranasally on Days 1 and 2 of life. On Day 3, pups were infected intranasally with PR-8 influenza, and survival was monitored. n = 15 per group from 3 independent experiments. (B) To test if LGG protected older infant mice, mouse pups were given 1.5 x106 Colony Forming Units of LGG intranasally on Day 6 of life. On Day 7, pups were infected intranasally with PR8 influenza, and survival was monitored. n = 14 per group from 3 independent experiments. To test if LGG conferred protection after influenza infection, mice were infected on the third day of life with influenza. (C) 24 hours or (D) 48 hours later, mice were given 1 x106 Colony Forming Units of LGG intranasally. n = 16 per group from 4 independent experiments. (E) Viral loads were determined at 3 and 6 days post infection by real time PCR. Data from 3 independent experiments. Real time PCR was performed to determine transcription of (F) IFNγ and (G) IL-6 at 3 days post-infection. Data from 4 independent experiments.
Fig 3
Fig 3. Differential recruitment of immune cells to the alveolar airspace in neonatal animals.
Mouse pups received 1 x106 Colony Forming Units of LGG intranasally on Days 1 and 2 of life. On Day 3, pups were infected intranasally with PR-8 influenza. (A) Histopathology was done at 3 and 6 days post-infection, which demonstrated similar bronchopulmonary infiltrates in both sham and LGG treated neonates. H&E Stain, representative figures. Arrows indicate areas of bronchopulmonary infiltration. Clinical severity scoring was performed (B). In separate experiments, cell counts for the specified cell types from (C) whole lung tissue and (D) bronchoalveolar lavage was determined by flow cytometry. Data from 3 independent experiments.
Fig 4
Fig 4. Lactobacillus causes differential transcriptional response to influenza.
Neonatal animals were given either LGG or sham treatment, and then infected with influenza on the third day of life. Animals were harvested 12 hours post-infection. Nanostring gene transcription analysis was performed on lungs. (A) Heatmap of all genes test in the LGG treated neonates, compared to expression in the other groups. Green: Upregulated; Brown: Down regulated. N = 3–4 per group, 2 experiments. (B) Venn diagram depicting the overlap of differentially regulated genes in the adult and the LGG-treated neonate. (C) IPA analysis of canonical pathways upregulated in the LGG-treated neonates. Significance P-values were calculated based on the Fisher's right tailed exact test. The log (p-value) are shown on the x-axis of the bar chart. The color of the bars indicates the activity (orange bars) or the inhibition (blue bars) of the predicted pathways. (D) IPA Top upstream regulators of gene expression in LGG-treated neonates versus uninfected neonates.
Fig 5
Fig 5. Sham-treated neonates begin to respond 24 hours post-infection.
LGG or sham treated animals were either mock or influenza infected on Day 3 of life. Animals were harvested 24 hours post-infection. Nanostring gene transcription analysis was performed on whole lungs. (A) Heatmap of all genes assayed in the LGG-treated neonates, compared to expression in all other groups. Green: Up regulated; Brown: Down regulated. N = 3 per group. (B) Principal component analysis of both 12 and 24 hours post infection samples.
Fig 6
Fig 6. LGG treatment alone is responsible for induction of inflammatory genes.
Neonatal animals were given either LGG or sham treatment, and then infected with influenza or sham on the Day 3 of life. Animals were harvested 24 hours post-infection. Nanostring gene transcription analysis was performed on lungs. MA plot of all genes tested at 24 hours post-infection, with (A) sham-treated influenza infected neonates compared to uninfected neonates; (B) LGG-treated uninfected neonates compared to uninfected neonates; (C) LGG-treated infected neonates compared to LGG-treated uninfected neonates; and (D) LGG-treated infected neonates compared to sham-treated infected neonates. Triangles denote statistical significance (p<0.05). Red: genes unique to infections; blue: genes unique to probiotic treatment; green: genes that are expressed by both probiotic and influenza infected animals independently; gold: interaction effect of LGG and influenza.
Fig 7
Fig 7. LGG promotes early transcription of Type I IFNs and survival of influenza virus-infected neonatal mice improves with IFNβ pretreatment.
Mouse pups received 1 x106 Colony Forming Units of LGG or sham intranasally on Day 1 of life and then harvested at various time points. Real time PCR was performed for (A) IFNα4 and (B) IFNβ1. N = 3–9 per time point, 3 experiments. (C) Low dose IFNβ or sham treatment was given on Days of 1 and 2 of life. Animals were infected with influenza virus and tracked for survival. Kaplan-Meier survival statistics were completed for all experiments. (n = 15 per group, 3 experiments).
Fig 8
Fig 8. Heat-killed LGG provides equivalent protection and Toll like receptor signaling is critical for the recognition of Lactobacillus.
(A) MyD88-/- and C57BL/6 pups received 1 x106 Colony Forming Units of LGG intranasally on Days 1 and 2 of life. On Day 3, pups were infected intranasally with PR-8 influenza, and survival was monitored. (p = 0.84, comparing the two MyD88-/- groups) (B) A549 cells were treated with indicated conditions and proteins were isolated for a western blot of p-SAPK/JNK. (C) Quantification of phosphorylation of pSAPK/JNK compared to untreated control. A similar treatment and infection protocol was followed for (D) Casp1-/- (E) TLR4-/-. N = 14–18 in each group from 4 independent experiments. (F) Mice were given 1 x108 Colony Forming Units of heat-killed LGG intranasally on Days 1 and 2 of life, and then infected with influenza on the third day of life; survival was monitored. n = 17 per group from 4 independent experiments.
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