Tumor necrosis factor signaling mediates resistance to mycobacteria by inhibiting bacterial growth and macrophage death

Abstract

Tumor necrosis factor (TNF), a key effector in controlling tuberculosis, is thought to exert protection by directing formation of granulomas, organized aggregates of macrophages and other immune cells. Loss of TNF signaling causes progression of tuberculosis in humans, and the increased mortality of Mycobacterium tuberculosis-infected mice is associated with disorganized necrotic granulomas, although the precise roles of TNF signaling preceding this endpoint remain undefined. We monitored transparent Mycobacterium marinum-infected zebrafish live to conduct a stepwise dissection of how TNF signaling operates in mycobacterial pathogenesis. We found that loss of TNF signaling caused increased mortality even when only innate immunity was operant. In the absence of TNF, intracellular bacterial growth and granuloma formation were accelerated and was followed by necrotic death of overladen macrophages and granuloma breakdown. Thus, TNF is not required for tuberculous granuloma formation, but maintains granuloma integrity indirectly by restricting mycobacterial growth within macrophages and preventing their necrosis.

Figure 1

TR1 morphant embryos are more susceptible to mycobacterial infection. (A) Control (Con) and TR1 morphant embryos were either mock injected (n=30 each) or infected with 108 +/- 11 colony forming units (CFU) of M. marinum on 1 dpf (n=50 each). Data are plotted as percentage of surviving embryos on each day. TR1 morphant embryos are significantly more susceptible to infection with M. marinum than controls (Hazard ratio 5.7, p < 0.0001, Kaplan Meier method with log-rank (Mantel-Cox) test). Survival was not statistically different between mock-injected TR1 and control embryos. (B) Mean bacterial loads per embryo for control (Con), TR1, and Pu.1 morphant embryos at 2, 4, and 6 days post injection (dpi) with 70 ± 13 CFU. p < 0.0001 by 1-way ANOVA. Data for Pu.1 embryos are only available for 2 and 4 dpi due to mortality. Error bars represent standard deviation from the mean. All morphant bacterial loads are significantly different from each other (p<0.05) at each individual time points (Control versus TR1, Control versus Pu.1, and TR1 versus Pu.1) by Student’s unpaired t-test. (C) Representative pictures of control, TR1, and Pu.1 morphant embryos at 4 dpi with fluorescence representing bacterial load. Scale bar, 500 μm.

Figure 2

Granulomas form faster in the absence of TNF signaling. (A) DIC (left) and fluorescence (right) images indicate activation of granuloma activated genes (gag3.13) in granulomas in both control and morphant embryos. Scale bars, 25 μm. (B) Three pools of ~30 embryos were injected with 56 ± 18 CFU of gag7 and four pools of ~30 embryos were injected with 64 ± 17 CFU of gag3.13. All groups were scored at four dpi for induction of gags as indicated by fluorescent activity. The average percentage of embryos with gag induction is plotted ± standard deviation of the mean. Averages for both gag7 and gag3.13 are significantly different between controls and morphants (p<0.05, Student’s unpaired t-test). (C-E) 23 Control and 14 TR1 morphant embryos were injected with 89 ± 4 CFU and followed sequentially for 5 dpi and monitored for granuloma formation and size. All dark bars represent control morphant data; light bars represent TR1 morphant data. Data is plotted as average ± standard error of the mean. (C) The percentage of embryos with at least one granuloma over time. TR1 morphant embryos have significantly more granulomas at 3 dpi (p<0.01) as analyzed by Fisher’s exact test of a contingency table. (D) The average number of granulomas identified by DIC and fluorescent microscopy over time. (E) TR1 morphant granulomas are significantly larger than control granulomas (p<0.05 at 3 dpi, p<0.0001 at 4 and 5 dpi by Student’s unpaired t-test, n=14 Control and 14 TR1 granulomas at 3 dpi, 75 control and 53 TR1 granulomas at 4 dpi, and 113 control and 72 granulomas at 5 dpi). Y axis represents granuloma diameter in μm. (F-G) Pools of 20 embryos were injected with 27 ± 6 wild-type (WT) and 51 ± 13 ΔRD1 M. marinum and assayed by microscopy at 4dpi for granuloma formation. (F) Representative images of control and TR1 morphant embryos. Arrowheads indicate granulomas. Scale bar, 200μm. (G) The average percentage of embryos with granulomas is plotted plus or minus standard deviation of the mean over four biological replicates. The percentage of embryos with granulomas is significantly higher when injected with WT (p<0.01 for Con; WT versus Con; ΔRD1 and p<0.001 for TR1; WT versus TR1; ΔRD1 by Student’s unpaired t-test).

Figure 3

Growth of Erp mutant bacteria is partially restored in TR1 morphant embryos. (A) Representative fluorescence images for control (Con) and TR1 morphant fish infected with 55 ± 7 CFU of Erp mutant (Erp) M. marinum at 4 dpi. Scale bar, 500 μm. (B-D) 50 individual macrophages were scored for multiplicity of infection (MOI) as either containing 10 or less bacteria (B) or more than 10 bacteria (C) at 3 dpi during infection with 55 ± 7 CFU of Erp mutant bacteria. Scale bar, 25 μm. (D) TR1 morphant embryos had a significantly higher percentage of macrophages with more than ten bacteria as compared to controls (p<0.05, Student’s unpaired t-test).

Figure 4

TR1 morphant embryos have significantly more acellular granulomas than controls. (A, B) Differential interference contrast overlay with fluorescence (left) of infected embryos then labeled using in situ hybridization for the macrophage marker fms (right). Green indicates M. marinum in fluorescent overlays. Purple staining of hybridized embryos indicates expression of the macrophage marker fms. (A) An example of a cellular granuloma in a control embryo before and after in situ hybridization indicating bacteria within aggregated macrophages. (B) An acellular granuloma example in a TR1 morphant embryo indicating bacteria found primarily outside of macrophages. (C) The results of a blind scoring of 82 granulomas in 22 WT embryos and 78 granulomas in 22 TR1 morphant embryos for cellularity indicates that TR1 granulomas were twice as likely to contain 1/3 or fewer fms-positive cells than to WT granulomas (p<0.0001) as analyzed by Fisher’s exact test of a contingency table. Embryos were 7 dpf and infected with 267 ± 28 CFU.

Figure 5

TNF signaling does not influence the rate of apoptosis of infected cells. DIC (A) and fluorescent (B-D) imaging of a granuloma. (B) Colocalization of TNF expression (red) and TUNEL-labeled double strand DNA breaks (green). Individual fluorescence channels for TNF expression (C) and TUNEL-labeling (D) are shown. Scale bar, 50 μm. (E) The average number of TUNEL-positive cells within granulomas in 4 dpi embryos is unchanged between control (n=16 granulomas) and TR1 morphant embryos (n=20). Infection of control embryos with RD1-deficient bacteria (ΔRD1) is used as a control (n=20). The number of TUNEL-positive cells in ΔRD1 granulomas is significantly less than control and TR1 morphant granulomas with wild-type bacteria (p<0.05 by Student’s unpaired t-test for both comparisons). Granulomas were selected to be between 40 and 50μm in diameter to normalize for total cell number. (F) The percentage of TUNEL-positive granulomas between control and TR1 morphant 4 dpi embryos is unchanged. Three separate experiments of pools of 20–40 granulomas were scored per condition and plotted as number of granulomas with TUNEL-positive cells over total granuloma number. Error bars represent standard error of the mean.

Figure 6

TR1 morphant embryos are more likely to have extracellular bacteria as indicated by cording. DIC (A) and fluorescence (B-E) imaging of bacteria in vivo. (AB) Extracellular bacteria in Pu.1 morphant embryos without macrophages displays the cording phenotype. Scale bar, 25 μm. (C-D) Flattened three-dimensional z-stacks of control (C) and TR1 morphant (D) granulomas demonstrate cording in TR1 morphant embryos. Scale bars, 50 μm. (E) Cording after what appears to be breakdown of an individual infected macrophage in infected TR1 morphant embryo. Scale bar, 25 μm. (G) Four pools of 30 embryos were injected with 18 ± 5 CFU and scored daily for the presence of extracellular bacteria as indicated by cording and plotted as the percentage of embryos with cording plus or minus the standard deviation. TR1 morphant embryos had significantly higher percentages of embryos with cording at all time points by Student’s unpaired t-test (p<0.05 for 3 and 4 dpi, p<0.01 for 5 dpi).

Figure 7

Conceptual overview of how TNF deficiency leads to changes during mycobacterial pathogenesis. TNF deficiency leads to increased bacterial growth and macrophage death, which in turn lead to the release of bacteria into the extracellular milieu and granuloma breakdown.