Mycobacteria control epithelial TLR responses.

<p>The impact of TLRs on mycobacterial modulated epithelial signalling was studied by Western blotting prior to infection and three days after infection. (a) Blocking of TLR2 (p = 0.0063) or TLR4 (p = 0.0047) prior to infection or stimulation with 19 kDa significantly increased epithelial pCREB production (p = 0.0163). (b) Blocking of TLRs or 19 kDa stimulation of epithelial cells had an non-significant impact on pGSK3βα expression. (c) Blocking of TLR2 or TLR4 before mycobacterial infection of primary epithelial cells non-significantly restored the NF-κB values to background levels. Data are presented as mean ± SEM of three separate experiments; *p<0.05 and **p<0.01.</p

Controlled epithelial cytokine secretion.

<p>Mycobacterial control of induced transcriptional factors was analysed as epithelial cytokine secretion from six hours up to three days after infection. Infection induced a significant (a) IL-6 and (b) IL-10 secretion that peaked at 72 hours (p = 0.0425 and p = 0.0186 compared to LPS). (c) Mycobacterial infection of primary epithelial cells induced an early significant IL-22 secretion (p = 0.0463 compared to LPS) that ended 24 hours after infection. Data are presented as mean ± SEM of four separate experiments; *p<0.05 and **p<0.01.</p

Mycobacteria bypass epithelial NF-κB signalling.

<p>(a) Infection of primary epithelial cells did not induce NF-κB activation quantified by ELISA, but an early activation of c-Jun proteins in epithelial cells was observed. (b) Epithelial GSK3αβ-pathway was analysed by Phospho-kinase array upon mycobacteria infection. In the beginning of infection, live mycobacteria, the virulence factors LAM and 19 kDa, and the TLR4 agonist LPS, induced comparable induction of p38, pAkt and pGSK3αβ. During the first 24 h, LPS induced higher increase of pCREB protein levels than mycobacteria (p = 0.0017). Third day of infection, mycobacteria significantly increased epithelial pCREB compared to medium control (p = 0.0357) or LPS (p = 0.0089). Epithelial stimulation with LAM induced an increase in pGSK3αβ and pAkt phosphorylation (p = 0.001 respectively p = 0.0196) during the later stages of infection compared to the early time-point. Generally, mycobacteria induced a more persistent increase of the investigated transcription factors three days after infection in primary epithelial cell than the controls LPS, 19 kDa and LAM. Data are presented as mean ± SEM of three separate experiments; **p<0.01 and ***p<0.001.</p

Mycobacteria modulate epithelial signalling pathways.

<p>Several molecules in the TLR-signalling pathway were analysed by Western blotting upon mycobacterial infection. (a–b) We could confirm that mycobacterial infection did not induce NF-κB- or IκB-activation. Mycobacterial suppression of primary epithelial (b) (p = 0.002) IκB and (d) (p = 0.0148) pGSK3αβ proteins were mostly pronounced at 24 hours of infection. The phosphorylated forms of (c) (p = 0.0163) CREB and (d) (p = 0.0248) GSK3αβ proteins reached highest levels 72 hours after infection. (e) Mycobacterial infection increased the Fos family of AP-1 proteins, as c-Fos protein levels significantly increased 72 hours after infection (p = 0.0038). (f) Mycobacteria induced two peaks of pERK1/2 protein levels, after 24 hours (p<0.001) and after 72 hours (p = 0.0034) of infection. (g) Epithelial cells express PPARγ protein, but mycobacterial infection did not significantly increase epithelial PPARγ amount. Data are presented as mean ± SEM of three experiments; *p<0.05, **p<0.01 and ***p<0.001.</p

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

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

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