Circulating neutrophil counts increase rapidly during invasive infection, while platelet counts decrease progressively over time.

<p>At 0, 5, 12, and 18 hours post infection with <i>S</i>. <i>pyogenes</i> the mice were sacrificed and a blood sample was taken by cardiac puncture. Flow cytometry of citrated blood was used to determine neutrophil (A) and platelet counts (B) in each animal. Horizontal line represents median, p-value calculated using a Mann-Whitney U test.</p

Platelet-Neutrophil complex formation is increased during the early stages of invasive infection and decreased during late stage infection.

<p>At 0, 5, 12, and 18 hours post infection with <i>S</i>. <i>pyogenes</i> the mice were sacrificed and a blood sample was taken by cardiac puncture. Flow cytometry of citrated blood was used to determine formation of platelet-neutrophil complexes in vivo (A) or post addition of thrombin ex vivo (B). Upregulation of CD11b on neutrophils was also determined (C). The median of the fluorescence for CD11b was measured for each sample with and without prior stimulation with fMLF ex-vivo. Stimulation with fMLF for each sample was set to 100% and the values displayed are for unstimulated samples. Horizontal line represents mean, p-value calculated using a t-test with Welch’s correction for unequal variances.</p

Platelets are progressively accumulated in the liver sinusoids at during invasive infection.

<p>At 0, 12, and 18 hours post <i>S</i>. <i>pyogenes</i> infection the mice were sacrificed and the liver was harvested, fixed and embedded in paraffin blocks, sectioned and immunohistochemistry was performed with anti CD41 to detect platelets. Representative images are shown and quantification of the number of platelet aggregates per slide is illustrated as a column graph, where each dot is an individual mouse.</p

Activated platelets are not found in ex-vivo blood samples during invasive infection however plasma levels of released CD62P are significantly increased over time.

<p>At 0, 5, 12, and 18 hours post infection with <i>S</i>. <i>pyogenes</i> the mice were sacrificed and a blood sample was taken by cardiac puncture. Flow cytometry of citrated blood was used to determine the activation status of the circulating platelet population activated platelets as median of the fluorescence (MFI) for CD62P (A) and JONA (B). Plasma was prepared and soluble CD62P was determined by ELISA (C). Horizontal line represents median, p-value calculated using a Mann-Whitney U test.</p

Organ damage increases over time during invasive infection and correlates with platelet activation and thrombocytopenia.

<p>At 0, 5, 12 and 18 hours post <i>S</i>. <i>pyogenes</i> infection the mice were sacrificed and a blood sample was taken by cardiac puncture. Plasma was prepared and AST activity was determined using a commercial kit (A). Horizontal line represents median, p-value calculated using a Mann-Whitney U test. Correlation between plasma AST activity and platelet count (B) or plasma AST activity and plasma CD62 levels (C) was performed using a Spearman’s rank correlation.</p

Weight loss and bacterial dissemination increases over time during invasive infection.

<p>Mice infected with <i>S</i>. <i>pyogenes</i> AP1 were weighed before infection and at 5, 12, and 18 hours post infection and the decrease in body weight was calculated (A). At 0, 5, 12, and 18 hours post infection the mice were sacrificed the bacterial load in blood (B), spleen (C), and liver (D) was determined by serial dilution of homogenates and viable count determination. Horizontal line (A) represents the mean, p-value calculated using a t-test. Horizontal line (B-D) represents median, p-value calculated using a Mann-Whitney U test.</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 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 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