Lactobacillus-mediated priming of the respiratory mucosa protects against lethal pneumovirus infection

Abstract

The inflammatory response to respiratory virus infection can be complex and refractory to standard therapy. Lactobacilli, when targeted to the respiratory epithelium, are highly effective at suppressing virus-induced inflammation and protecting against lethal disease. Specifically, wild-type mice primed via intranasal inoculation with live or heat-inactivated Lactobacillus plantarum or Lactobacillus reuteri were completely protected against lethal infection with the virulent rodent pathogen, pneumonia virus of mice; significant protection (60% survival) persisted for at least 13 wk. Protection was not unique to Lactobacillus species, and it was also observed in response to priming with nonpathogenic Gram-positive Listeria innocua. Priming with live lactobacilli resulted in diminished granulocyte recruitment, diminished expression of multiple proinflammatory cytokines (CXCL10, CXCL1, CCL2, and TNF), and reduced virus recovery, although we have demonstrated clearly that absolute virus titer does not predict clinical outcome. Lactobacillus priming also resulted in prolonged survival and protection against the lethal sequelae of pneumonia virus of mice infection in MyD88 gene-deleted (MyD88(-/-)) mice, suggesting that the protective mechanisms may be TLR-independent. Most intriguing, virus recovery and cytokine expression patterns in Lactobacillus-primed MyD88(-/-) mice were indistinguishable from those observed in control-primed MyD88(-/-) counterparts. In summary, we have identified and characterized an effective Lactobacillus-mediated innate immune shield, which may ultimately serve as critical and long-term protection against infection in the absence of specific antiviral vaccines.

Figure 1. Priming of the respiratory mucosa with live lactobacilli results in protection from an otherwise lethal virus infection

A. Standard experimental protocol. On days -14 and -7, BALB/c mice were inoculated intranasally with 10⁹ cfu L. plantarum (LP), 10⁹ cfu L. reuteri (LR), or PBS + 1% BSA vehicle control (PBS). On day 0, all mice were inoculated with pneumonia virus of mice (PVM; 2000 virion copies/μL). B. Survival of mice inoculated as indicated with LP, LR or PBS prior to virus infection, n = 10 mice per group, representative of at least two independent experiments; ***p < 0.001 C. Granulocytes detected in bronchoalveolar lavage (BAL) fluid at time points indicated (day -6, day -4, day -1 as per Fig. 1A; n = 5 – 7 mice per time point). Horizontal bars indicate mean values; statistical significance **p < 0.01; ***p < 0.001. D. Survival of mice primed with LP or PBS days -14 and -7 and challenged with PVM 13 weeks (91 days) later; n = 10 mice per group, **p < 0.01.

Figure 2. Priming of the respiratory mucosa with heat-inactivated lactobacilli and other gram-positive bacteria

Standard experimental protocol is as per Fig. 1A unless otherwise noted. A. Survival of mice inoculated with LP, heat-inactivated (hi) LP (10⁹ cfu-equivalents) or PBS prior to virus infection; B. Survival of mice inoculated with 10⁹ cfu Listeria innocua (LI), 10⁹ cfu-equivalents heat-inactivated L. innocua (LI), or PBS prior to virus infection; C. Survival of naïve BALB/c mice inoculated with PVM on day 0, then inoculated with lactobacilli (10⁹ cfu LP, 10⁹ cfu-equivalents heat-inactivated (hi) LP, or PBS + BSA) on day 3. Each group includes 10 or more mice per condition, representative of at least two independent experiments ***p < 0.001.

Figure 3. Virus recovery from lung tissue of Lactobacillus- and control-primed mice

A. Virus recovery from lung tissue at days 3, 5 and 7 after inoculation with PVM from mice treated with either Lactobacillus spp. or PBS + BSA control, as determined by qRT-PCR with two standard curves [Suppl. Fig. 1]; n = 5 – 7 mice per timepoint per condition, *p < 0.05; **p < 0.01; n.s., not significant. B. and C. Survival of and virus recovery from Lactobacillus (LP) and PBS + BSA-primed mice. The virus (PVM) inoculum received by ten PBS + BSA -primed mice was reduced to 600 copies/μL so that peak recovery at day 5 would be comparable to that detected in the ten LP-primed mice. Virus recovery was evaluated in 5 mice selected randomly on day 5 from each group as shown, and % survival of 5 mice remaining is as shown; **p < 0.01.

Figure 4. Histopathologic analysis

Hematoxylin and eosin-(H&E) stained lung tissue from mice primed with PBS + BSA (A., B., and C.) or L. plantarum in PBS + BSA (D., E., and F.) prior to virus infection; shown here at day 7 as per Fig. 1A. A. and B. Diffuse alveolar, bronchiolar, and perivascular inflammation is observed in lung tissue from PVM-infected, control-primed mice (original magnifications, 5X, and 10X, respectively). C. Arrows indicate infiltrating granulocytes (original magnification, 40X) seen exiting from the blood vessel in the center of the field in panel B. D. and E. Lung tissue from L. plantarum-primed, PVM-infected mice exhibits diminished alveolar inflammation and pronounced peribronchiolar and perivascular cuffing (original magnifications, 5X and 10X, respectively). F. Enlarged from E. (at arrow) dense lymphocyte-enriched inflammatory infiltrate, consistent with descriptions of induced bronchus-associated lymphoid tissue (iBALT; original magnification, 40X.)

Figure 5. Flow cytometric analysis of leukocyte subsets

A. Flow cytometric analysis of whole-lung single-cell suspensions generated from control-(PBS + BSA) or L. plantarum (LP)-primed, PVM-infected mice (day 7, n = 5 – 6 mice per condition). Total lung cells were gated for side-scatter (SSC) and expression of the cell surface granulocyte marker GR1, shown with no-antibody and isotype-matched antibody controls. Data shown are representative of four independent experiments. B. Percentage of total viable lung cells identified as granulocytes (GR1⁺) or lymphocytes (identified by characteristic forward/side scatter) from PBS + BSA-primed, PVM-infected or L. plantarum-primed, PVM-infected mice. C. Percentage of total viable lymphocytes (in B.), with CD4⁺ T cell (CD3⁺CD4⁺CD8⁻), CD8⁺ T cell (CD3⁺CD4⁻CD8⁺) NK cell (CD3⁻DX5⁺) or B lymphocyte (B220⁺) immunophenotype; * = p < 0.05; ** = p < 0.01. Data shown are compiled from four independent experiments.

Figure 6. Lactobacillus-mediated priming of the respiratory mucosa leads to suppression of multiple virus-induced proinflammatory cytokines

A. Expression of transcripts encoding proinflammatory mediators in lungs of Lactobacillus- or PBS-primed, PVM-infected mice, normalized to expression in Lactobacillus- or PBS-primed, uninfected mice, respectively (day 7 as per Fig. 1A, n = 4 mice per experimental group; data compiled from three independent experiments). B. Immunoreactive CXCL10, CCL2, CXCL1, and TNF detected in BAL fluid of Lactobacillus- or PBS-primed, PVM-infected mice; C. Immunoreactive IFN-β and IL-10 detected in BAL fluid of Lactobacillus- or PBS-primed, PVM-infected mice; n = 4 – 5 mice per experiment; data compiled from three independent experiments; * p < 0.05; ** p < 0.01; ***p < 0.001; LP, L. plantarum; LR, L. reuteri.

Figure 7. Lactobacillus-primed MyD88 gene-deleted (MyD88^−/−) mice are also protected against the lethal sequelae of PVM infection

A. Survival of C57BL/6 mice primed with 10⁹ cfu L. plantarum or PBS + BSA prior to PVM infection; n = 20 mice per group. B. Survival of MyD88^−/− mice primed with 10⁹ cfu L. plantarum or PBS + 1% BSA control prior to PVM infection; n = 8 – 10 mice per group. Data shown are representative of three independent experiments, ** = p < 0.01; LP = L. plantarum. C. Virus recovery from lung tissue determined by qRT-PCR of L. plantarum-primed and PBS-primed C57BL/6 and MyD88^−/− mice on day 7 after PVM infection (as per Fig. 1A); n = 5 – 8 mice per timepoint per condition, *p < 0.01; data compiled from two independent experiments.

Figure 8. Lactobacillus-priming of MyD88^−/− mice has no impact on virus-mediated production of inflammatory cytokines

Immunoreactive A. CCL2, B. CXCL1, C. CXCL10, D. IL-10, and E. TNF detected in lung tissue homogenates from C57BL/6 and MyD88^−/− mice at day 7 after virus infection; n = 5 – 8 mice per time point per condition, *p < 0.05; **p < 0.01; data compiled from two independent experiments.