Cording Mycobacterium tuberculosis Bacilli Have a Key Role in the Progression towards Active Tuberculosis, Which is Stopped by Previous Immune Response

Abstract

Cording was the first virulence factor identified in Mycobacterium tuberculosis (Mtb). We aimed to ascertain its role in the induction of active tuberculosis (TB) in the mouse strain C3HeB/FeJ by testing the immunopathogenic capacity of the H37Rv strain. We have obtained two batches of the same strain by stopping their growth in Proskauer Beck liquid medium once the mid-log phase was reached, in the noncording Mtb (NCMtb) batch, and two days later in the cording Mtb (CMtb) batch, when cording could be detected by microscopic analysis. Mice were challenged with each batch intravenously and followed-up for 24 days. CMtb caused a significant increase in the bacillary load at an early stage post-challenge (day 17), when a granulomatous response started, generating exudative lesions characterized by neutrophilic infiltration, which promoted extracellular bacillary growth together with cording formation, as shown for the first time in vivo. In contrast, NCMtb experienced slight or no bacillary growth and lesions could barely be detected. Previous Bacillus Calmette-Guérin (BCG) vaccination or low dose aerosol (LDA) Mtb infection were able to delay the progression towards active TB after CMtb challenge. While BCG vaccination also reduced bacillary load when NCMtb was challenged, LDA did not, and its proliferative lesions experienced neutrophil infiltration. Analysis of lung cytokine and chemokine profiles points to their capacity to block the production of CXCL-1 and further amplification of IL-1β, IL-17 and neutrophilic extracellular trap formation, all of which are essential for TB progression. These data highlight the key role of cording formation in the induction of active TB.

Keywords: BCG; C3HeB/FeJ mice; CXCL-1; Mycobacterium tuberculosis; cording; neutrophilic extracellular traps; neutrophils.

Figure 1

Impact of cording bacilli challenge on the progression towards active tuberculosis (TB). (A) Aggregate area distribution pattern for both batches. Each column represents the frequency of spots for each area interval, logarithmically distributed between 1 and 10⁴ μm². (B) Bacillary load (BL) and (C) damaged lung area progression after intravenous (IV) challenge with cording Mtb(CMtb) or non-cording Mtb(NCMtb).

Figure 2

Induction of cording in vivo. SEM micrographs of exudative lesions showing in vivo cording formation. Lower amplification showing one of the representative lesions found in lungs from CMtb mouse. (A) One of the zones explored in depth in the lungs is marked with a red circle. Formation of cording bacilli is marked with a white ellipse (B) or white arrows (C,D). (E) The formation neutrophilic extracellular traps (NETs). (F) Detail with trapped bacilli. Scale bars equal 100 μm in (A) and 1 μm in the others.

Figure 3

Protective effect of BCG immunization. (A) Progression of pulmonary BL, (B) lung damaged area and (C) evolution of the lesions. Each circle represents an animal (A) or a recut (B) and lines are medians. Asterisks indicate differences within the same batch; bar and asterisks indicate differences between batches. Mann–Whitney test (* p < 0.05, ** p < 0.01). Scale bars equal 1 mm and asterisks point lung lesions.

Figure 4

Impact of BCG vaccination on the immune response in lungs. Analysis of inflammatory mediators in lung homogenates at days 17, 21 and 24 PI. Results are represented as log10 of the concentration in pg/mL. Each circle represents an animal and lines are medians. Asterisks indicate differences within the same batch; bar and asterisks indicate differences between batches. Mann–Whitney test (* p < 0.05, ** p < 0.01).

Figure 5

Protective effect of low dose aerosol (LDA) infection. (A) Progression of pulmonary BL, (B) lung damaged area (C) and evolution of the lesions. Each circle represents an animal (A) or a recut (B) and lines are medians. Asterisks indicate differences within the same batch; bar and asterisks indicate differences between batches. Mann–Whitney test (* p < 0.05, ** p < 0.01). Scale bars equal 1 mm and asterisks point lung lesions.

Figure 6

Impact of LDA infection in the immune response in lungs. Analysis of inflammatory mediators in lung homogenates at days 17, 21 and 24 PI. Results are represented as log10 of the concentration in pg/mL. Each circle represents an animal and lines are medians. Asterisks indicate differences within the same batch; bar and asterisks indicate differences between batches. Mann-Whitney test (* p < 0.05, ** p < 0.01).

Figure 7

Comparison of PCA of inflammatory mediators after BCG vaccination (A) or LDA infection (B) in lung homogenates. PCA biplot showing each animal and variable (top). PC1 scores for each animal, lines are means; ANOVA and Sidak’s multiple comparisons test (** p < 0.01, *** p < 0.001) (bottom left). Mediators’ contribution to PC1 (bottom right).