Bacille Calmette-Guérin (BCG)-associated inflammation and fibrosis: modulation by recombinant BCG expressing interferon-gamma (IFN-gamma)

Abstract

Immunization with existing BCG vaccines has failed to confer consistent protection against tuberculosis. One of the ways to improve the efficacy of BCG is by enhancing its ability to induce a type-1 T cell response. However, this approach carries the risk that enhanced immunoreactivity may exacerbate tissue pathology associated with vaccination. The aim of the present study was to determine whether use of a recombinant BCG expressing IFN-gamma (BCG-IFN) would result in an alteration in the pattern of inflammation and local tissue fibrosis. A murine intravenous BCG infection model was used in which there was a time- and dose-dependent increase in the weight and number of granulomas in the liver. Infection was associated with increased inflammatory activity in the liver, as shown by the increase in expression of inducible nitric oxide synthase (iNOS) assessed by immunochemistry and by measurement of specific mRNA, and in fibrosis measured by hydroxyproline content of the liver and percentage of granuloma cells staining positively for type 1 procollagen. Infection with BCG-IFN resulted in a reduction in organ weight and bacterial load on day 21 compared with infection with control BCG transformed with vector alone (BCG-plasmid). By day 21, there was also a reduction in iNOS mRNA and iNOS+ cells in granulomas in mice infected with BCG-IFN compared with infection with BCG-plasmid, and a similar reduction in both total number of granulomas and liver hydroxyproline content. These results demonstrate that the granulomas in the areas of mycobacterial infection are active sites of both inflammation and fibrosis, and that the local expression of IFN-gamma by the recombinant BCG results in more efficient bacterial clearance which is accompanied by a reduction in tissue pathology.

Fig. 1

(a) Percentage of cells positive for inducible nitric oxide synthase (iNOS) by immunochemistry in granulomas of liver on different days post-infection in BALB/c mice infected with BCG. (Results represent mean of six mice ± s.e.m.) (b) Abundance of mRNA expression for iNOS by reverse transcriptase-polymerase chain reaction in liver on different days post-infection in BALB/c mice infected with BCG. Lanes 1, 2 and 3 represent three representative samples from each group.

Fig. 2

Amount of hydroxyproline in liver (a) and percentage of cells positive for type I procollagen by immunochemistry in granulomas of liver (b) on different days post-infection in BALB/c mice infected with BCG. (Results represent mean of six mice ± s.e.m.)

Fig. 3

(a) Weight of spleen and liver as percentage of body weight in mice infected with 2 × 10⁶ BCG transformed with empty plasmid (BCG-plasmid; ▪) or BCG expressing IFN-γ (BCG-IFN; □) on different days post-infection. Results represent mean of 6–12 mice ± s.e.m. in two separate experiments at each day post-infection; *P < 0.05 on day 21 in spleen and liver. (b) BCG colony-forming units (CFU) in mice infected with 2 × 10⁶ BCG carrying only plasmid (BCG-plasmid; ▪) or BCG expressing IFN-γ (BCG-IFN; □) on different days post-infection. Results represent mean of 6–12 mice ± s.e.m. in two separate experiments at each day post-infection; *P < 0.05 on day 21 in spleen and liver. (c) Number of granulomas (i), amount of hydroxyproline in liver (ii) and percentage of cells positive for procollagen 1 in liver granulomas (iii) in mice infected with 2 × 10⁶ BCG transformed with empty plasmid (BCG-plasmid; ▪) or BCG expressing IFN-γ (BCG-IFN; □) on day 21 post-infection. Results represent mean of 12 mice; *P < 0.05.

Fig. 4

(a) Comparison of abundance of mRNA expression for inducible nitric oxide synthase (iNOS), IFN-γ and tumour necrosis factor-alpha (TNF-α) as ratio of β-actin in liver of mice infected with BCG transformed with empty plasmid (BCG-plasmid; ▪) or BCG expressing IFN-γ (BCG-IFN; □) on day 21 post-infection. *P < 0.05. (b) Comparison of immunoscoring for iNOS⁺ cells in granulomas of liver in mice injected with BCG transformed with empty plasmid (BCG-plasmid; ▪) or BCG expressing IFN-γ (BCG-IFN; □) on day 21 post-infection. *P < 0.05.