Adjunctive etanercept reduces TB-associated pathology.

<p>Hematoxylin and eosin staining (20x and 200x – inset) was performed on lung tissues. Images shown are representative of sections obtained from 4 animals per group. Each time-point is shown in weeks on the individual images. Lung pathology in animals receiving adjunctive etanercept (RHZ plus etanercept) (panel B) appeared to resolve earlier than those receiving standard treatment (RHZ alone) (panel A). Morphometric analysis confirmed these findings and demonstrated reduced lung involvement in mice treated with adjunctive etanercept (RHZ plus etanercept; blue x) versus standard treatment (RHZ alone; red □) (panel C). Results are represented as percentage lung involvement, calculated using ImageJ software.</p

Bacterial burden in the lungs of mice.

<p>Six weeks after an aerosol infection with <i>Mycobacterium tuberculosis</i>, C3HeB/FeJ mice were split among four treatment groups: Untreated (no treatment), standard TB treatment (RHZ), etanercept alone and standard TB treatment with adjunctive etanercept (RHZ + Etanercept). The number of viable bacteria in the lungs were estimated by determining colony-forming units (CFU). Results are shown for the duration of study (panel A) and also as individual dot plots for 8 (panel B) and 10 (panel C) weeks after starting TB treatment. Compared to standard TB treatment, the addition of etanercept resulted in a significantly lower pulmonary bacterial burden, corresponding to the phase when a significant proportion of bacteria are multiplying slowly (panels B, C). Results are presented as mean (±SD) CFU in the lungs, detected from a minimum of four mice at each time point and for each group. CFU are presented on a logarithmic scale (log₁₀) in panel A and on a linear scale for panels B and C and represent the same data.</p

Immune responses during TB treatments.

<p>Splenic recall assays were performed to monitor the immune responses during TB treatment in animals receiving adjunctive etanercept (RHZ plus etanercept) (blue x) and standard treatment (RHZ alone) (red □). Infected but untreated mice (black ○) and mice treated with etanercept alone (green Δ) were used as controls. High levels of IL-2, IFN-γ and CCL4 were noted in mice receiving standard TB treatment with or without adjunctive etanercept. Conversely, higher levels of IL-10 were only noted in the untreated mice. Overall, this represents a transition, from a regulatory to an effector phenotype, following the onset of treatment. Determinations were made from four mice, and each assay was performed in triplicate. Results are expressed as mean level (pg/ml) (±SD).</p

<i>M. tuberculosis</i> PknD-specific IgG in vaccinated guinea pigs.

<p><i>M. tuberculosis</i>, blood was harvested from each animal to obtain sera. The levels of IgG antibodies reactive to heat-inactivated <i>M. tuberculosis</i> or to recombinant PknD sensor were determined for each group. While sera from BCG-vaccinated animals demonstrated high levels of IgG antibodies reactive to heat-inactivated <i>M. tuberculosis</i> (P<0.01) (panel A), only sera from PknD-vaccinated animals showed high levels of IgG antibodies reactive to PknD (P<0.01) (panel B). Sera from three animals from each group were tested. Each assay was performed in triplicate. Data are presented on a linear scale as mean±standard deviation.</p

PknD vaccination protects against <i>M.</i><i>tuberculosis</i> dissemination to the brain.

<p>Colony-forming units (CFU) in the lungs (panel A) and brains (panel B) of guinea pigs vaccinated with BCG (triangle), recombinant PknD (inverted triangle), DDA (adjuvant alone) (square) or PBS (unvaccinated controls) (circle), 4- and 6-weeks after an aerosol challenge with <i>M. tuberculosis</i> are shown. BCG vaccination limited the pulmonary bacillary load (P = 0.01) and also significantly reduced the bacillary burden in the brains after aerosol challenge with virulent <i>M. tuberculosis</i> (P = 0.01). While PknD vaccination did not limit bacillary growth in the lungs, it offered significant protection against bacillary dissemination to the brain, which was no different from BCG (P>0.24), even though the PknD vaccinated animals had almost 100-fold higher bacterial burdens in the lungs. Four guinea pigs per group were sacrificed at each time-point, except for the DDA group, for which only three animals were sacrificed 6-weeks after aerosol challenge. Data are presented on a logarithmic scale as mean±standard deviation.</p

PknD vaccination induces specific IFN-γ and IgG responses.

<p>Splenic recall assays were performed on splenocytes from guinea pigs vaccinated with BCG, recombinant PknD, DDA (adjuvant alone) or PBS (unvaccinated controls). The WST-1 system was then used to measure proliferation after stimulation heat-inactivated <i>M. tuberculosis</i> (white bars), recombinant PknD subunit (striped bars) or control with culture media alone (black bars) (panel A). Significant cellular proliferation in response to PknD subunit was only observed in splenocytes from PknD vaccinated animals (P = 0.002). Similarly, significant proliferation in response to heat-inactivated <i>M. tuberculosis</i> was only observed in splenocytes from BCG vaccinated animals (P = 0.01). Supernatants from these assays were also used to measure the IFN-γ levels (panel B). Consistent with proliferation data, only animals vaccinated with PknD (P<0.01) generated appreciable levels of IFN-γ in response to the PknD subunit protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066310#pone-0066310-g002" target="_blank">Figure 2B</a>). Splenocytes from three animals from each group were tested. Each assay was performed in triplicate. Data are presented on a linear scale as mean±standard deviation.</p

Pre-incubation of <i>M.</i><i>tuberculosis</i> with sera from PknD-vaccinated guinea pigs reduces invasion of brain microvascular endothelia:

<p>Human brain microvascular endothelia are the primary components of the blood-brain barrier and protect the CNS from the systemic circulation. Brain microvascular endothelial invasion of <i>M. tuberculosis</i> pre-incubated with sera from each vaccinated group normalized to the unvaccinated animals (PBS) is shown. While sera from the BCG vaccinated animals was not protective, pre-incubation of <i>M. tuberculosis</i> with sera from PknD-vaccinated guinea pigs significantly reduced (>10 fold) invasion of brain microvascular endothelia (P<0.01). Sera from three animals from each group were tested. Each assay was performed in triplicate. Data are presented on a linear scale as mean±standard deviation.</p