Figure 1.
TNF-α activity in the lungs of treated mice.
TNF-α activity was measured in lung homogenates obtained from Mycobacterium tuberculosis infected C3HeB/FeJ mice by WEHI assay (A) and ELISA (B). Levels for mice receiving no treatment (Untreated), standard TB treatment (RHZ) or with adjunctive etanercept (RHZ + Etanercept) were measured 7 days after the start of treatment. TNF-α activity increased with TB treatment and was abrogated by the administration of adjunctive etanercept. Moreover, to ensure that the TNF-α activity measured using the WEHI assay was indeed due to TNF-α and not other endogenous factors, lung homogenates from both the standard treatment (RHZ alone) and adjunctive etanercept (RHZ plus etanercept) arms were pre-incubated with increasing concentrations of a murine anti-TNF-α antibody and tested using the WEHI assay (C). Lung homogenates from the adjunctive etanercept arm (open bars) had consistently lower TNF-α activity which remained unaffected by pre-incubation with the neutralizing antibody. However, TNF-α activity in the lung homogenates from the standard treatment arm (filled bars) decreased in a dose-dependent manner with increasing concentrations of the neutralizing antibody, with activities at 100 or 10 fold dilutions reaching levels observed in the adjunctive etanercept arm. Analyses were performed on two-fold diluted lung homogenate from four mice per group. Data is normalized to the total protein concentration of the tissue samples and is presented as mean activity (U/ml/g) (±SD) in panels A, C and mean level (pg/ml) (±SD) in panel B.
Figure 2.
Bacterial burden in the lungs of mice.
Six weeks after an aerosol infection with Mycobacterium tuberculosis, 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.
Figure 3.
Adjunctive etanercept reduces TB-associated pathology.
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.
Figure 4.
Immune responses during TB treatments.
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).