Characterisation and development of histopathological lesions in a guinea pig model of Mycobacterium tuberculosis infection

Abstract

Tuberculosis (TB) remains a very significant infectious disease worldwide. New vaccines and therapies are needed, even more crucially with the increase of multi-drug resistant Mycobacterium tuberculosis strains. Preclinical animal models are very valuable for the development of these new disease control strategies. Guinea pigs are one of the best models of TB, sharing many features with the pathology observed in human TB. Here we describe the development of TB lesions in a guinea pig model of infection. We characterise the granulomatous lesions in four developmental stages (I-IV), using histopathological analysis and immunohistochemical (IHC) techniques to study macrophages, T cells, B cells and granulocytes. The granulomas in the guinea pigs start as aggregations of macrophages and few heterophils, evolving to larger lesions showing central caseous necrosis with mineralisation and abundant acid-fast bacilli, surrounded by a rim of macrophages and lymphocytes in the outer layers of the granuloma. Multinucleated giant cells are very rare and fibrotic capsules are not formed in this animal model.

Keywords: granuloma; guinea pig; immunohistochemistry, cell marker, Mycobacterium tuberculosis; pathology, animal model; tuberculosis.

Figure 1

Representative microphotographs of the four stages of tuberculous granulomas in lungs from guinea pigs (H&E). (A, B) Stage I. Small with epithelioid macrophages (black arrows) with interspersed lymphocytes and heterophils (red arrows). (C, D) Stage II. Organised granulomas with heterophils (red arrows) in the centre and surrounded by epithelioid macrophages interspersed with polymorphonuclear cells (black arrows). (E, F) Stage III. Organised, with initial to caseous necrosis with degenerated and/or viable heterophils (red arrows). (G, H) Stage IV. Organised, advanced tuberculous lesion with extensive caseous necrosis and mineralisation. Scale bars = 250 μm (insets B, D, F, and H = 100 μm).

Figure 2

Representative microphotographs of multinucleated giant cells (MNGCs) (H&E) and Ziehl-Neelsen (ZN) staining. (A) Epithelioid macrophages forming an initial MNGC (arrows) in a stage I granuloma at 4 wpi and (B) several MNGCs (arrows) in a stage II granuloma at 8 wpi. (C) Acid-fast bacilli (AFBs, arrows) in lungs of guinea pigs with stage II (4 wpi) and (D) stage III (24 wpi) tuberculous granuloma (ZN staining). Scale bars A and B = 100 μm (insets, and C and D = 50 μm).

Figure 3

Representative microphotographs of Martius Scarlet Blue (MSB; blue for collagen) staining in lungs of guinea pigs with tuberculous granulomas. (A) Stage IV granuloma within the lung parenchyma, not showing a fibrotic capsule surrounding the lesion. (B) Stage IV granuloma located between an artery (A) and a bronchiole (B) showing normal collagen (blue stain) from the lung around the granuloma. Scale bars = 250 μm.

Figure 4

Number of different granulomas from different developmental stages (I–IV) observed in guinea pig lungs at different time points after inoculation (from 5 dpi to 24–27 wpi). Each dot represents a lung section and bars represent median values.

Figure 5

Number of viable Mtb H37Rv in lung at day 5 and 10, and weeks, 3, 4, and 8 weeks post-infection by the aerosol route of delivery. Bacterial load in lungs at 24–27 weeks post-infection represent the result of relapse of disease following sub-optimal delivery of drug treatment delivered between weeks 3–7 post-infection. Individual dots represent each animal in each group. Viable bacterial load are expressed as Log₁₀ cfu/mL, each animal in the group represented as black circles, and group mean expressed as horizontal black bars, in violin plots.

Figure 6

Iba1+ immunohistochemical detection in stage I, II, III, and IV granulomas throughout the experiment. (A) Percentage of Iba1⁺ staining in stage I to IV granulomas at 3, 4, 8, and 24–27 wpi. Dots show the individual percentage of staining in each granuloma. Lines show the median value of the percentage of staining in the different stages throughout the study. (B) Iba1 expression in stage I granuloma at 8 wpi. (C) Iba1 expression in stage II granuloma at 8 wpi. (D) Iba1 expression in stage III granuloma at 4 wpi. (E) Iba1 expression in stage IV granuloma at 8 wpi. Insets show close-up images at higher magnification showing Iba1⁺ macrophages. Scale bars = 500 μm (insets = 100 μm).

Figure 7

MAC387+ immunohistochemical detection in stage I, II, III, and IV granulomas throughout the experiment. (A) Percentage of MAC387⁺ cells in stage I to IV granulomas at 3, 4, 8, and 24–27 wpi. Dots show the individual percentage of staining in each granuloma. Lines show the median value of the percentage of staining in the different stages throughout the study. (B) MAC387 expression in stage I granuloma at 4 wpi. (C) MAC387 expression in stage II granuloma at 4 wpi. (D) MAC387 expression surrounding a necrotic core (arrowhead) in stage III granuloma at 4 wpi. (E) MAC387 expression in stage IV granuloma at 8 wpi. Insets show close-up images at higher magnification showing MAC387⁺ cells: macrophages and polymorphonuclear cells. Scale bars = 500 μm (insets = 100 μm).

Figure 8

Myeloperoxidase (MPO) + immunohistochemical detection in stage I, II, III, and IV granulomas throughout the experiment. (A) Percentage of MPO⁺ staining in stage I to IV granulomas at 3, 4, 8, and 24–27 wpi. Dots show the individual percentage of staining in each granuloma. Lines show the median value of the percentage of staining in the different stages throughout the study. (B) MPO expression in stage I granuloma at 4 wpi. (C) MPO expression in stage II granuloma at 4 wpi. (D) MPO expression in stage III granuloma at 4 wpi. (E) MPO expression in stage IV granuloma at 8 wpi. Insets show close-up images at higher magnification showing MPO⁺ polymorphonuclear cells. Scale bars = 500 μm (insets = 100 μm).

Figure 9

B-cell Specific Activator Protein+ (SAP+) immunohistochemical detection in stage I, II, III, and IV granulomas throughout the experiment. (A) Percentage of B-cell SAP+ staining in all stages granulomas at 3, 4, 8, and 24–27 wpi. Dots show the individual percentage of staining in each granuloma. Lines show the median value of the percentage of staining in the different stages throughout the study. (B) B-cell SAP expression in stage I granuloma at 8 wpi. (C) B-cell SAP expression in stage II granuloma at 8 wpi. (D) B-cell SAP expression in stage III granuloma at 4 wpi. (E) B-cell SAP expression in stage IV granuloma at 8 wpi. Insets show close-up images at higher magnification showing B cell SAP⁺ lymphocytes. Scale bars = 500 μm (insets = 100 μm).

Figure 10

CD3+ immunohistochemical detection in stage I, II, III, and IV granulomas throughout the experiment. (A) Percentage of CD3⁺ staining in stage I to IV granulomas at 3 to 24–27 wpi. Dots show the individual percentage of staining in each granuloma. Lines show the median value of the percentage of staining in the different stages throughout the study. (B) CD3 expression in stage I granuloma (arrowheads) at 24–27 wpi. (C) CD3 expression in stage II granuloma at 24–27 wpi. (D) CD3 expression in stage III granuloma (arrowheads) at 24–27 wpi. (E) CD3 expression in stage IV granuloma at 8 wpi. Insets show close-up images at higher magnification showing CD3+ T lymphocytes. Scale bars = 500 μm (insets = 100 μm).