BCG induces protection against Mycobacterium tuberculosis infection in the Wistar rat model

Abstract

Our understanding of the correlation of Mycobacterium bovis Bacille Calmette-Guerin (BCG)-mediated immune responses and protection against Mycobacterium tuberculosis (Mtb) infection is still limited. We have recently characterized a Wistar rat model of experimental tuberculosis (TB). In the present study, we evaluated the efficacy of BCG vaccination in this model. Upon Mtb challenge, BCG vaccinated rats controlled growth of the bacilli earlier than unvaccinated rats. Histopathology analysis of infected lungs demonstrated a reduced number of granulomatous lesions and lower parenchymal inflammation in vaccinated animals. Vaccine-mediated protection correlated with the rapid accumulation of antigen specific CD4(+) and CD8(+) T cells in the infected lungs. Immunohistochemistry further revealed higher number of CD8(+) cells in the pulmonary granulomas of vaccinated animals. Evaluation of pulmonary immune responses in vaccinated and Mtb infected rats by real time PCR at day 15 post-challenge showed reduced expression of genes responsible for negative regulation of Th1 immune responses. Thus, early protection observed in BCG vaccinated rats correlated with a similarly timed shift of immunity towards the Th1 type response. Our data support the importance of (i) the Th1-Th2 balance in the control of mycobacterial infection and (ii) the value of the Wistar rats in understanding the biology of TB.

Figure 1. BCG vaccination of Wistar rats leads to protection early during infection.

A) BCG vaccinated rats (N = 5) were challenged 6 weeks post vaccination with ∼100 CFU of Mtb W4 strain and the bacillary load in the lungs was evaluated till 120 days post-challenge and compared with that of age-matched unvaccinated rats. The difference in Mtb load between BCG vaccinated and unvaccinated group over time is significant (P<0.005, ANOVA). B) BCG vaccinated rats were challenged after 8 weeks of vaccination (N = 6) and infection was followed for 15 days. C) BCG vaccinated rats were challenged with Mtb after 6 weeks of vaccination and lung bacilli load at 120 days post-challenge was estimated and compared to that of unvaccinated rats. Values are means ± SDs (*, P<0.05; **, P<0.005; ***, P<0.0005; Mann-Whitney test).

Figure 2. BCG vaccination of Wistar rat results in reduction in granulomatous inflammation in lung.

A) Representative histopathologic micrographs of lungs of BCG vaccinated and unvaccinated rats challenged with Mtb W4 sacrificed at day 60 post-challenge. H&E, magnification 2.5× and 20×. BCG vaccinated rats have reduced number of granulomas and more normal lung parenchyma. B) Number of granulomas observed in 10 different lung sections per animal (N = 5 per group) at day 60 post-challenge. Values are means ± SDs (*, P<0.05).

Figure 3. BCG vaccinated rats has more CD8⁺ cells in the lung granulomas.

Representative micrographs of immunohistochemical staining of CD8⁺ cells in the lungs of BCG vaccinated and unvaccinated rats challenged with M. tuberculosis W4 and sacrificed at day 30 post-challenge, magnification 40×. Some of the positive cells have been indicated by an arrow.

Figure 4. Specific T cell responses in BCG-vaccinated and Mtb challenged rats.

Wistar rats were vaccinated with BCG. After 6 weeks vaccinated and age-matched unvaccinated rats were challenged with ∼100 CFU of Mtb W4. After 15, 30, 60 and 120 days post-challenge lung cells were isolated and exposed ex vivo to heat-killed Mtb and were analyzed for IFN-γ production as described in Materials and Methods. A) Total numbers of mycobacteria specific IFN-γ⁺CD4⁺ T cells in lung at the indicated days after Mtb challenge. B) Total numbers of mycobacteria specific IFN-γ⁺CD8⁺ T cells in lung at the indicated day after Mtb challenge. C) Amount of IFN-γ produced by lung cells upon ex vivo stimulation. Bars represent mean ± SD for cells from five individually analyzed rats (*, P<0.05, Student's t test).

Figure 5. Quantitative PCR of RNA from lung cells demonstrates a decrease in negative regulators of Th1 immunity in Mtb challenged vaccinated rats.

A) Total RNA from lung cells of Mtb challenged BCG vaccinated and unvaccinated rats (N = 5) was isolated at 15 days post-challenge and the relative expression levels for all 84 genes in the two groups are plotted against each other in the scatter plot. Genes encoding Socs1, Irf4, NF-κβ1 and JunB are down-regulated by at least two-fold in BCG vaccinated rats relative to unvaccinated rats (green circles). B) Cluster-diagram showing differential expression pattern of four of the above mentioned genes in individual rat belonging to the two treatment groups.

Figure 6. Hypothetical model of the regulation of IFN-αβ and IL-4 signaling in BCG vaccinated Mtb infected rats.

Upon infection Beijing strains induce IFN-αβ . BCG vaccination in Wistar rat inhibits the genes involved in IFN-αβ and IL-4 signaling and thus down regulates the negative regulators of Th1 immunity (indicated by red arrows). Downregulated genes observed in this study (Table S1) have been indicated in blue.