Extensive tissue necrosis and oedema formation in mice lacking IFNγ.

<p>HE stained histologic sections of foot pads from representative WT (A) and IFNγ^-/- (B) mice 1, 5 and 8 weeks after infection with <i>M</i>. <i>ulcerans</i>. 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).</p

IFNγ-deficient mice have a significantly higher bacterial burden 5 weeks after infection.

<p>WT and IFNγ^-/- mice were infected with <i>M</i>. <i>ulcerans</i> 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 <i>M</i>. <i>ulcerans</i> (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).</p

Faster progression of <i>M</i>. <i>ulcerans</i> infection in IFNγ-deficient mice.

<p>WT and IFNγ^-/- mice were infected into the left hind foot pad with <i>M</i>. <i>ulcerans</i> 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 <i>M</i>. <i>ulcerans</i> 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.</p

Absence of specific antibody responses against <i>M</i>. <i>ulcerans</i> in infected WT and IFNγ^-/- mice.

<p>Sera of WT and IFNγ^-/- mice were analyzed 5 and 8 weeks after infection for the presence of specific IgG antibody responses against <i>M</i>. <i>ulcerans</i> by Western blotting on <i>M</i>. <i>ulcerans</i> whole cell lysate. A monoclonal antibody specific for the <i>M</i>. <i>ulcerans</i> antigen MUL3720 served as positive control.</p

Antibodies for immunohistochemistry on pig skin.

<p>Antibodies for immunohistochemistry on pig skin.</p

Local Cellular Immune Responses and Pathogenesis of Buruli Ulcer Lesions in the Experimental <i>Mycobacterium Ulcerans</i> Pig Infection Model

<div><p>Background</p><p>Buruli ulcer is a neglected tropical disease of the skin that is caused by infection with <i>Mycobacterium ulcerans</i>. We recently established an experimental pig (<i>Sus scrofa</i>) infection model for Buruli ulcer to investigate host-pathogen interactions, the efficacy of candidate vaccines and of new treatment options.</p><p>Methodology/Principal Findings</p><p>Here we have used the model to study pathogenesis and early host-pathogen interactions in the affected porcine skin upon infection with mycolactone-producing and non-producing <i>M</i>. <i>ulcerans</i> strains. Histopathological analyses of nodular lesions in the porcine skin revealed that six weeks after infection with wild-type <i>M</i>. <i>ulcerans</i> bacteria extracellular acid fast bacilli were surrounded by distinct layers of neutrophils, macrophages and lymphocytes. Upon ulceration, the necrotic tissue containing the major bacterial burden was sloughing off, leading to the loss of most of the mycobacteria. Compared to wild-type <i>M</i>. <i>ulcerans</i> bacteria, toxin-deficient mutants caused an increased granulomatous cellular infiltration without massive tissue necrosis, and only smaller clusters of acid fast bacilli.</p><p>Conclusions/Significance</p><p>In summary, the present study shows that the pathogenesis and early immune response to <i>M</i>. <i>ulcerans</i> infection in the pig is very well reflecting BU disease in humans, making the pig infection model an excellent tool for the profiling of new therapeutic and prophylactic interventions.</p></div

Different histopathological appearance of lesions caused by mycolactone producing and non-producing <i>M</i>. <i>ulcerans</i> strains.

<p>Histological sections of a nodular lesion six weeks after infection with 1.3 x 10⁵ CFU wild-type <i>M</i>. <i>ulcerans</i> (A). The organisation of cellular infiltration around a necrotic core containing AFB (A5) and fat cell ghosts (A7) was comparable to lesions caused by higher doses of <i>M</i>. <i>ulcerans</i>. The central part of the nodule contained neutrophils (A1, A2), surrounded by a ring of macrophages (A3, A4) that was interspersed with T-cells (A6). Infection with 3.1 x 10⁵ CFU of mycolactone-deficient <i>M</i>. <i>ulcerans</i> led to the development of a lesion with more inhomogeneously organized infiltration (B). Several granulomatous structures contained small cores consisting of neutrophils (B1, B2) and AFB (B5). Macrophage infiltration was massive (B4) and interspersed with numerous T-cells (B6). Fat cell ghosts were absent and necrosis was less extensive (B7).</p

Layers of leukocyte infiltration in nodular lesions six weeks after infection.

<p>Histological sections of a nodular lesion six weeks after infection with 1.3 x 10⁶ CFU of <i>M</i>. <i>ulcerans</i>. Ziehl-Neelsen/Methyleneblue (ZN) staining revealed a strong cellular infiltration around a central necrotic core containing AFB stained in pink (A1, A2). The necrotic core and the surrounding ring of infiltration consisted of neutrophils (B1, B2). Neutrophils were surrounded by a belt of macrophages (C1, C2) that were heavily interspersed with T-cells (D1, D2).</p

Epidermal hyperplasia above nodular lesions.

<p>Histological sections of nodular lesions stained with Haematoxylin/Eosin. (A) Small nodule caused by infection with 1.3 x 10⁵ CFU <i>M</i>. <i>ulcerans</i>. (B) Large nodule caused by infection with 1.3 x 10⁶ CFU <i>M</i>. <i>ulcerans</i>. Epidermal hyperplasia was strongest directly above the lesion (central box) and weaker if further away from the lesion.</p

Established immunohistochemical stainings for leukocytes in the pig skin.

<p>Serial histologic sections of pig skin stained with Haematoxylin/Eosin (HE), Ziehl-Neelsen/Methyleneblue (ZN) and by immunohistochemistry protocols established for antibodies specific for T-cells (CD3), macrophages (IBA-1, CD107a) or neutrophils (21 kDa neutrophil protein). A B-cell cluster is shown that can only be identified by exclusion criteria (asterisk). The cluster of cells (asterisk) was not stained positive with antibodies against T-cells (CD3), macrophages (IBA-1, CD107a) nor neutrophils (Neutrophils), while the cells have lymphocyte appearance (ZN, insert).</p