Innate Immune Responses after Airway Epithelial Stimulation with <i>Mycobacterium bovis</i> Bacille-Calmette Guérin

<div><p><i>Mycobacterium bovis</i> bacilli Calmette-Guerin (BCG) is used as a benchmark to compare the immunogenicity of new vaccines against tuberculosis. This live vaccine is administered intradermal, but several new studies show that changing the route to mucosal immunisation represents an improved strategy. We analysed the immunomodulatory functions of BCG on human neutrophils and primary airway epithelial cells (AECs), as the early events of mucosal immune activation are unclear. Neutrophils and the primary epithelial cells were found to express the IL-17A receptor subunit IL-17RA, while the expression of IL-17RE was only observed on epithelial cells. BCG stimulation specifically reduced neutrophil IL-17RA and epithelial IL-17RE expression. BCG induced neutrophil extracellular traps (NETs), but did not have an effect on apoptosis as measured by transcription factor forkhead box O3 (FOXO3). BCG stimulation of AECs induced CXCL8 secretion and neutrophil endothelial passage towards infected epithelia. Infected epithelial cells and neutrophils were not found to be a source of IL-17 cytokines or the interstitial collagenase MMP-1. However, the addition of IFNγ or IL-17A to BCG stimulated primary epithelial cells increased epithelial IL-6 secretion, while the presence of IFNγ reduced neutrophil recruitment. Using our model of mucosal infection we revealed that BCG induces selective mucosal innate immune responses that could lead to induction of vaccine-mediated protection of the lung.</p></div

Production of Neutrophil extracellular traps (NETs) during BCG infection.

<p>Primary neutrophils were stimulated with BCG for one, two and three hours in the presence of IL-17A and/or IFN-γ. (A) NETs were studied in a light microscope after 2 hours and stained with DAPI and (B) large NET structures were observed in addition to single NETs. (C) Quantification of NETs was performed with Picogreen assay on cell supernatants after 3 hours and (D) over time at 1, 2 and 3 hours. Results are depicted as mean ± SD from a total of 4 donors. Means of samples were were compared with negative control using ANOVA followed by Dunnet’s multiple comparisons test and significance was accepted at *p < 0.05, **p< 0.01, or ***p < 0.001. Bars represent 25 μm (A, B left panel) or 100 μm (B, right panel)</p

IL-17RA and IL-17RE expression on primary epithelial cells and neutrophils.

<p>IL-17RA and IL-17RE expression was assessed by flow cytometry in (A) primary AECs and (B) neutrophils. The results are depicted as mean ± SD of median fluorescence intensity (MFI) of a total of six (epithelial cells) or five (neutrophils) experiments. In addition, representative histograms are shown for each experiment. Abbreviations: secondary antibody control (neg), IgG control (IgG) or receptor expression on cells (IL-17RA/RE). Comparisons were performed with Mann-Whitney test and significance was accepted at *p < 0.05, **p< 0.01, or ***p < 0.001.</p

The effect of BCG infection on epithelial cytokine expression.

<p>(A) CXCL8 and (B) IL-6 production from epithelial cells stimulated with BCG in combination with IL-17A and/or IFN-γ for 0, 24, 48 and 72 hours, MOI 1:1. The 72 hours time points are in addition represented as bar graphs for (C) CXCL8 and (D) IL-6. The results are depicted as mean ± SD of three experiments. Means were compared with one way ANOVA followed by multiple comparisons test with Tukey’s correction and significance was accepted at *p < 0.05, **p< 0.01, or ***p < 0.001.</p

Neutrophil migration during BCG infection.

<p>(A) Epithelial cells were seeded to the bottom well of a transwell system and stimulated with BCG for 3 days, MOI 1:1. After infection, primary neutrophils were added to a layer of HUVEC cells in the top insert well. Neutrophil transmigration over the membrane was measured by counting the number of neutrophils in the bottom well compared to the insert after 3 hours. Results are depicted as mean ± SEM percentage of neutrophil diapedesis. Means were compared with students t-test and significance was accepted at *p < 0.05, **p< 0.01, or ***p < 0.001.</p