Fig 1.
Faster progression of M. ulcerans infection in IFNγ-deficient mice.
WT and IFNγ-/- mice were infected into the left hind foot pad with M. ulcerans and the progression of the disease was followed by weekly measurements of the foot pad thickness (A) and documented with pictures of the infected feet (B). (A) IFNγ-/- mice exhibited an accelerated progression of M. ulcerans infection. At weeks 5 and 6, the foot pad thickness was significantly higher in IFNγ-/- mice than in WT animals. Mean values of the foot pad thickness (mm) are shown, the error bars represent the S.D. P values were calculated using non-parametric Mann-Whitney test. ****, P ≤ 0.0001; *, P ≤ 0.05; n.s., not significant. (B) Pictures of representative feet taken 1, 3, 5 and 8 weeks after infection. At week 5, all mice deficient for IFNγ-/- had swollen feet. No swelling was observed in WT mice at this time point. Eight weeks after infection, foot pad swelling was observed for both WT and IFNγ-/- mice but the macroscopic disease symptoms were more severe in mice lacking IFNγ. Scale bars represent 5 mm.
Fig 2.
Extensive tissue necrosis and oedema formation in mice lacking IFNγ.
HE stained histologic sections of foot pads from representative WT (A) and IFNγ-/- (B) mice 1, 5 and 8 weeks after infection with M. ulcerans. Scale bars represent 5 mm (A1, A4, A7, B1, B4 and B7, left), 1 mm (A1, A4, A7, B1, B4 and B7, box), 150 μm (A2, A5, A8, B2, B5 and B8) and 80 μm (A3, A6, A9, B3, B6 and B9).
Fig 3.
IFNγ-deficient mice have a significantly higher bacterial burden 5 weeks after infection.
WT and IFNγ-/- mice were infected with M. ulcerans and the bacterial load was determined by IS2404-specific qPCR (A). The distribution of AFB in the footpads was assessed by histopathological analysis at week 3 (B) and week 5 (C). (A) IFNγ-/- mice showed a significantly stronger increase in the bacterial burden between week 3 and 5 (1) and had a significantly higher bacterial burden as compared to WT animals 5 weeks after infection with M. ulcerans (2). Values are displayed as mean, the error bars represent the S.D. (n = 3 per genotype). P values were calculated using non-parametric regression models according to the Brunner-Langer method. *, P ≤ 0.05. (B and C) 5 μm tissue sections of foot pads from representative WT (left) and IFNγ-/- (right) mice stained with ZN for visualization of AFB after 3 (B) or 5 (C) weeks of infection. AFB were predominantly intracellular at week 3 (B1 and B2, black arrows) whereas a mix of intra- and extracellular bacilli was found after 5 weeks of infection (C). At week 5, more AFB were present in IFNγ-/- foot pads (C2 and C4), no difference in the total immune cell infiltration between the two groups was observed (C1 and C3). Scale bars represent 8 μm (B1 and B2), 160 μm (C1 and C3) and 40 μm (C2 and C4).
Fig 4.
Absence of specific antibody responses against M. ulcerans in infected WT and IFNγ-/- mice.
Sera of WT and IFNγ-/- mice were analyzed 5 and 8 weeks after infection for the presence of specific IgG antibody responses against M. ulcerans by Western blotting on M. ulcerans whole cell lysate. A monoclonal antibody specific for the M. ulcerans antigen MUL3720 served as positive control.