Transgenic mice enriched in omega-3 fatty acids are more susceptible to pulmonary tuberculosis: impaired resistance to tuberculosis in fat-1 mice

Abstract

BACKGROUND. Besides their health benefits, dietary omega-3 fatty acids (n-3 PUFAs) can impair host resistance to intracellular pathogens. Previously, we and others have showed that n-3 PUFA-treated macrophages poorly control Mycobacterium tuberculosis infection in vitro. METHODS. Wild-type and fat-1 transgenic mice were infected with virulent H37Rv M. tuberculosis via the aerosol route. We evaluated bacteriological and histopathological changes in lungs, as well as differences in activation and antimycobacterial capacity in primary macrophages ex vivo. RESULTS. fat-1 mice were more susceptible to tuberculosis, as demonstrated by higher bacterial loads and less robust inflammatory responses in lungs. Macrophages obtained from fat-1 mice were more readily infected with M. tuberculosis in vitro, compared with wild-type macrophages. This impaired bacterial control in cells from fat-1 mice correlated with reduced proinflammatory cytokine secretion, impaired oxidative metabolism, and diminished M. tuberculosis-lysotracker colocalization within phagosomes. CONCLUSIONS. We showed that endogenous production of n-3 PUFAs in fat-1 mice increases their susceptibility to tuberculosis, which could be explained in part by diminished activation and antimycobacterial responses in cells from fat-1 mice. These data suggest that n-3 PUFA-supplemented diets might have a detrimental effect on immunity to M. tuberculosis and raise concerns regarding the safety of omega-3 dietary supplementation in humans.

Figure 1

Increased bacterial loads in fat-1 mice. Animals were infected with Mycobacterium tuberculosis via aerosol infection. At 1 day and at 2, 4, 8, and 12 weeks, mice were euthanized, and the numbers of viable bacteria in lung and spleen were determined. Bacterial counts were estimated by the colony forming unit (CFU) assay, as described in the Materials and Methods. Data show bacterial survival as log₁₀ CFU (mean ± standard error of the mean) in fat-1 (dashed line) versus wild-type (WT) mice (solid line). *P < .005; **P < .05; ***P < .01.

Figure 2

Representative hematoxylin and eosin–stained lung sections from fat-1 (panels b, d, f, and h) and wild-type (WT; panels a, c, e, and g) mice after infection with Mycobacterium tuberculosis. The total magnification is ×200. Animals were infected with M. tuberculosis via aerosol infection. At 1 day and at 2, 4, and 8 weeks mice were euthanized, and histological changes in lung and spleen were evaluated as described in the Materials and Methods.

Figure 3

Increased mycobacterial permisiveness in fat-1 macrophages. Cells were infected with green fluorescent protein (GFP)-expressing Mycobacterium tuberculosis for 1 h, and the relative percentage of M. tuberculosis–infected cells was quantified by fluorescent microscopy, as described in the Materials and Methods. Representative fluorescent images of the increased bacterial accumulation in fat-1 cells (A) at 1 h postinfection, compared with wild-type (WT) cells (B). Arrows show infected cells. Data are representative of 3 independent experiments. C, Quantitative data represent the percentage of infected cells. D, Bacterial counts were estimated at different time points by the colony forming unit (CFU) assay, as described in the Materials and Methods. Data show bacterial survival as log₁₀ CFU (mean ± standard error of the mean; n = 10). *P < .001.

Figure 4

Reduced cytokine production in infected fat-1 macrophages. TNF-α (A), interleukin-6 (B), interleukin-1β (C), and monocyte chemotactic protein–1 (D) protein concentrations were quantified in culture supernatants at various times following infection with virulent Mycobacterium tuberculosis H37Rv by enzyme-linked immunosorbent assay, as described in the Materials and Methods. *P < .001; **P < .05; ***P < .01

Figure 5

Acquisition of lysotracker by green fluorescent protein (GFP) Mycobacterium tuberculosis–containing phagosomes and reactive oxygen generation was reduced in infected fat-1 macrophages. Phagosomal maturation was defined based on lysotracker colocalization and visualized by fluorescent microscopy, as described in the Materials and Methods. A, Representative images of the reduced colocalization in fat-1 macrophages, compared with wild-type (WT) macrophages. Data are representative of 3 independent experiments. B, Quantitative data show the percentage of lysotracker-positive mycobacterial phagosomes in fat-1 and WT cells. Reactive oxygen intermediates were estimated by FACS, as described in the Materials and Methods. C, Representative density plots of reduced respiratory burst in infected fat-1 macrophages. The analysis was restricted to the cell population in density plots and the region R2 was created for events with high intensity of red dihydroethidium (EB) fluorescence. The percentage of events in that region was determined by CellQuest. The basal level of oxidized EB due to metabolic activity of control uninfected cells was used to set gates. Data are representative of 3 independent experiments. D, Quantitative data show the relative percentage of EB red fluorescence. *P < .001; **P < .01.