Table 1.
Index case and HHC data of Mtb strains selected for mouse infections.
Fig 1.
Comparison of bacterial burden in the lungs and peripheral organs of C3HeB/FeJ mice infected with Mtb-HT and Mtb-LT strains.
Mice were infected with a low dose inoculum (50–120 CFU) with the three Mtb-HT and three Mtb-LT strains. 5 mice were included for each time point. At indicated time points following aerosol infection, lungs were harvested, and serial dilutions of lung homogenates were plated on 7H11 agar plates and the bacterial load was determined between 28–35 days of incubation at 37°C. Two-way ANOVA showed significant difference between animals infected with HT and LT groups. Week 2: LT-1 vs HT-1, HT-3 (p < 0.01); week 4: LT-2 vs HT-1, HT-2, HT-3, LT-3 (p < 0.0001); week 6: LT-2 vs HT-1, HT-2, HT-3, LT-3 (p < 0.0001); week 12: LT-1 vs HT-1, HT-2, HT-3, LT-3 (p < 0.05).
Fig 2.
Increased inflammatory response and cellular infiltration in Mtb-LT infected mice.
Single cell preparations were obtained, and trypan blue exclusion method was used to calculate the total live cells in the lungs of Mtb-HT and Mtb-LT infected C3HeB/FeJ mice at 4 weeks post-infection (A). Lung cells from 4 week-infected mice were surface stained with directly conjugated antibodies to evaluate the total number of immune cell subsets by flow cytometry (B). Immunostaining with anti-Ly6G performed on paraffin-embedded fixed lung tissue sections from Mtb-HT and Mtb-LT infected mice. The image is a representative of three animals from each of the six infections. (C). For A and B, data are from five mice and presented as mean +/- SEM. One-way Anova was used to compare HT-1 infection with the rest of the groups.
Fig 3.
Differential granulomatous response in C3HeB/FeJ mice infected with Mtb-HT and Mtb-LT strains.
Mosaic H&E images of lung tissue representing acute (2–4 weeks) and chronic (10–12 weeks) phases of infection from the three Mtb-HT infections (A) and three Mtb-LT infections (B). Mosaic images were created using Surveyor software with Turboscan by objective imaging at 20X. Each group includes 4–5 mice per time point. Each time point includes representative images from all three Mtb-HT and Mtb-LT infections.
Fig 4.
Mtb-HT infected C3HeB/FeJ mice form caseating granulomas.
Representative histological image of murine lung infected with Mtb-HT1 during chronic infection, show distinctive fibrotic and necrotic lesions (A). The zoomed in area demonstrates the extent of inflammation observed in these granulomas. Presence of necrotic debris in the center core (yellow arrow), foamy macrophages (cyan arrow) and surrounding layer of fibroblasts (dashed arrow) and is seen in the granulomas (B). Solid arrow points to outer edge of granuloma fusing with an airway (C). Representative image of AFB staining at 12 weeks post infection (D). Serial sections were stained using Trichrome staining and show collagen deposition (seen in blue) on the outer periphery of the granuloma (E). Leica SCN400 F whole-slide scanner was used for scanning histological sections up to 40X magnification and images were analyzed using Aperio ImageScope.
Fig 5.
Mice infected with Mtb-HT strains show presence of solid discrete granulomas.
H&E stained sections of formalin-fixed paraffin-embedded tissues from the lungs of Mtb-HT infected C3HeB/FeJ mice obtained at 12 weeks post infection show well defined tubercle granuloma (square box) (A). Higher magnifications zoomed-in images show presence of peribronchiolar lymphocytes (B) and open alveolar spaces with less inflamed alveolar septae (C). AFB staining (D). High magnification images were taken using a Leica DM6000B by objective imaging at 20X and 100X and also by analyzing high-magnification scanned images using Aperio ImageScope. These images are representative of pathology seen with all three Mtb-HT infections.
Fig 6.
Infiltrative pathology in the lungs of Mtb-LT-infected C3HeB/FeJ.
H&E stained sections of formalin-fixed paraffin-embedded tissues from the lung of Mtb-LT infected C3HeB/FeJ mice obtained at 12 weeks post infection (A). Zoomed in area showing necrotic areas with karyorrhectic debris with alveolar space “A”, lymphocytic cluster “L” and neutrophilic debris “N” (B), and cellular exudate in airways (C). High magnification images were taken using a Leica DM6000B by objective imaging at 20X and 100X also by analyzing high-magnification scanned images using Aperio ImageScope. Ziehl-Neelsen acid-fast staining (D). These images are representative of pathology seen with all three Mtb-LT infections.
Table 2.
Type of granuloma development in Mtb-HT and Mtb-LT infected mice between 12–16 weeks following infection.
Fig 7.
Increased growth of Mtb-LT strains in macrophages is linked to increased lipid droplet accumulation.
MH-S cells were infected in triplicate with Mtb-HT and Mtb-LT strains at an MOI of 10. A total of 10 Mtb-HT and 10 Mtb-LT strains were analyzed, and it included the three Mtb-HT and three Mtb-LT strains described previously. Two days post-infection, cells were fixed in 4% paraformaldehyde, following which they were incubated with HCS LipidTOX Red dye. Mean Fluorescence intensity (MFI) is represented for LipidTOX Red. The average data for each strain is presented as a dot and the cumulative data as interquartile range with median. The data are representative of 1 of 2 individual experiments. Unpaired t-test was used to calculate the significant differences between cells infected with Mtb-HT and Mtb-LT strains (**p<0.01) (A). MH-S cells were grown on coverslips, infected with either Mtb-HT1 or Mtb-LT1. 48 hours later cells were processed for confocal imaging. The data is a representative confocal imaging showing cell nuclei stained with DAPI (blue) and presence of neutral lipids detected through HCS LipidTOX Red dye (B). BMDMs were differentiated for 7 days in D-10 containing 20% L-cell. Cells were infected using 3 MOI of the indicated strains (5 replicate wells for each condition). Data presented are day 7 CFU with and without MPN (final concentration of 100nM) (C) and are representative of one of three individual experiments. Data is represented as mean +/- SEM and significant differences in intracellular bacterial growth were calculated using one way ANOVA (*p <0.05 and ****p <0.0001).