Lactobacillus priming of the respiratory tract: Heterologous immunity and protection against lethal pneumovirus infection

Abstract

We showed previously that wild-type mice primed via intranasal inoculation with live or heat-inactivated Lactobacillus species were fully (100%) protected against the lethal sequelae of infection with the virulent pathogen, pneumonia virus of mice (PVM), a response that is associated with diminished expression of proinflammatory cytokines and diminished virus recovery. We show here that 40% of the mice primed with live Lactobacillus survived when PVM challenge was delayed for 5months. This robust and sustained resistance to PVM infection resulting from prior interaction with an otherwise unrelated microbe is a profound example of heterologous immunity. We undertook the present study in order to understand the nature and unique features of this response. We found that intranasal inoculation with L. reuteri elicited rapid, transient neutrophil recruitment in association with proinflammatory mediators (CXCL1, CCL3, CCL2, CXCL10, TNF-alpha and IL-17A) but not Th1 cytokines. IFNγ does not contribute to survival promoted by Lactobacillus-priming. Live L. reuteri detected in lung tissue underwent rapid clearance, and was undetectable at 24h after inoculation. In contrast, L. reuteri peptidoglycan (PGN) and L. reuteri genomic DNA (gDNA) were detected at 24 and 48h after inoculation, respectively. In contrast to live bacteria, intranasal inoculation with isolated L. reuteri gDNA elicited no neutrophil recruitment, had minimal impact on virus recovery and virus-associated production of CCL3, and provided no protection against the negative sequelae of virus infection. Isolated PGN elicited neutrophil recruitment and proinflammatory cytokines but did not promote sustained survival in response to subsequent PVM infection. Overall, further evaluation of the responses leading to Lactobacillus-mediated heterologous immunity may provide insight into novel antiviral preventive modalities.

Figure 1. Priming of the respiratory mucosa with live Lactobacillus species protects mice from lethal sequelae of subsequent virus challenge

A. Standard experimental protocol and timeline. Mice are inoculated intranasally with Lactobacillus (Lac), either 10⁹ cfu live Lactobacillus plantarum, 10⁸ cfu live Lactobacillus reuteri, or PBS/BSA (PBS) control on day -14 and again on day -7. On day 0 or at a time point thereafter, mice are challenged with pneumonia virus of mice (PVM). B. Survival of 8 week old BALB/c mice inoculated with L. plantarum as indicated in A., and challenged with PVM on day +153 (5 months), n = 10 mice per group. C. Survival of 8 week old BALB/c mice primed on day -14 and day -7 with PBS/BSA, with PBS/BSA on day -14 and L. plantarum on day -7, with L. plantarum on day -14 and PBS/BSA on day -7, or with L. plantarum on day -7 and on day -14; all inoculated with PVM on day +14, n = 5 mice/group. D. Virus recovery on day +17 (PVM_SH/GAPDH) from lung tissue of mice primed and challenged with PVM as in (C.) E. Survival of 3 week old C57BL/6 mice primed on day -14 and day -7 with PBS/BSA only, with L.reuteri on day -14 and PBS/BSA on day -7, with PBS/BSA on day -14 and L. reuteri on day -7, or L. reuteri on day -7 and day -14; n = 10 mice/group; statistical significance, *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 2. Proinflammatory cytokines detected in response to Lactobacillus priming

A. CXCL1, B. CCL3, C. CCL2, D. CXCL10, E. TNF-α and F. IL-17A detected in whole lung homogenates (pg / mL / mg lung tissue) at three time points after the first (day -14) and second (day -7) intranasal inoculation with L. reuteri (Lac, filled bars) or PBS/BSA control (PBS, open bars); n = 4 – 8 mice/time point, *p < 0.05, **p < 0.01, ***p < 0.005. No type I interferons were detected at any time point. Data compiled from 2 independent experiments.

Figure 3. Lactobacillus-priming and interferon-γ

A. IFNγ detected in whole lung homogenates (pg/mL /mg lung tissue) at three time points after first (day -14) and second (day -7) intranasal inoculation with L. reuteri (Lac, filled bars) or PBS/BSA control (PBS, open bars); n = 4 – 8 mice / time point. B. Survival of IFNγ^−/− and wild-type mice inoculated with L. plantarum as per standard protocol (Fig. 1A) and challenged at day 0 with PVM; *p < 0.05; **p < 0.01.

Figure 4. Neutrophils are recruited to the lungs in response to Lactobacillus priming

Cells from whole lung tissue evaluated by flow cytometry at three time points after the first (at day -14) and second (at day -7) inoculations with L. reuteri (Lac; filled bars) or PBS/BSA (PBS; open bars); A. Total lung cells B. Percent Gr1⁺ (neutrophils); n = 3 mice pooled for each time point, data compiled from 3 – 5 independent experiments; *p < 0.05; C. & D. Formalin-fixed, hematoxylin and eosin (H&E)-stained lung tissue from day -6, after second L. reuteri inoculation (see Fig. 1A); original magnifications, 10X and 40X, respectively.

Figure 5. Differential leukocyte recruitment in response to Lactobacillus priming

Cells from whole lung tissue evaluated by flow cytometry as in Fig. 4. A. Percent CD3+ lymphocytes B. percent CD11c⁺MHCII^hi dendritic cells; p < 0.05.

Figure 6. Lactobacillus clearance from lung tissue

A. Live L. reuteri detected in lung tissue at time points after inoculation with 10⁸ cfu; n = 3 mice/time point. B. L. reuteri genomic DNA (gDNA) detected by qPCR targeting 16S–23S intergenic region, normalized to a standard curve. C. L. reuteri PGN detected by kinetic melanin-generating spectrophotometric assay (see Suppl. Fig. 1) normalized to S. aureus standards; *p < 0.05; **p < 0.01; ***p < 0.005.

Figure 7. Priming with L. reuteri genomic DNA does not elicit protection from the lethal sequelae of PVM infection

A. Cells from whole lung tissue evaluated by flow cytometry after a first (day -14) and second (day -7) inoculation with 150 µg L. reuteri gDNA or diluent (dH₂O). Percent neutrophils (Gr1⁺ cells) at each time point are as shown; n = 3 mice per time point per condition, data compiled from 3 experiments. B. Virus recovery (PVM_SH /GAPDH) from mice primed with 50 µg L. reuteri genomic DNA or diluent (dH₂0) at days +3 and +6 after PVM challenge on day 0 C. Detection of CCL3 / MIP-1α in whole lung homogenates from mice primed and PVM-challenged as in B. D. Survival of mice inoculated with L. reuteri gDNA (50 or 150 µg at days -7 and -14) or dH₂O diluent control, and challenged with PVM on day 0, n = 5 mice per group;

Figure 8. Priming with peptidoglycan results in neutrophil recruitment and proinflammatory cytokine production but does not protect mice against the lethal sequelae of PVM infection

A. Percentage of GR1⁺ neutrophils detected in single cell suspensions from whole lung from mice inoculated with 100 µg PGN compared to 10⁸ cfu L. reuteri (Lac). B. – F. Proinflammatory cytokines CXCL1, CCL3, CCL2, TNF-α, and CXCL10 detected in whole lung homogenates after first (day -14) and second (day - 7) intranasal inoculations with 100 µg PGN (filled symbols) or PBS/BSA control (open symbols); G. Survival of mice primed with PGN (100 µg / mouse; filled symbols) or PBS/BSA (PBS; open symbols) prior to inoculation with PVM on day 0; n = 10 mice/group; statistical significance, *p < 0.05.