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. 2017 Nov 17;8(8):1918-1926.
doi: 10.1080/21505594.2017.1370530. Epub 2017 Oct 5.

Infiltrating leukocytes surround early Buruli ulcer lesions, but are unable to reach the mycolactone producing mycobacteria

Marie-Thérèse Ruf  1   2 , Christina Steffen  3 , Miriam Bolz  1   2 , Peter Schmid  1   2 , Gerd Pluschke  1   2
Affiliations

Infiltrating leukocytes surround early Buruli ulcer lesions, but are unable to reach the mycolactone producing mycobacteria

Marie-Thérèse Ruf et al. Virulence. .
No abstract available

Keywords: buruli ulcer; early pathogenesis; histopathology; infiltration; mycobacterium ulcerans; mycolactone.

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Figures

Figure 1.
Figure 1.
Histopathological overview over 5 of the early ulcerative lesions. Sections were either stained with Haematoxylin-Eosin (HE; A, D, G, J, M) or Ziehl-Neelsen/Methyleneblue (ZN-M; B, C, E, F, H, I, K, L, N, O) according to the WHO standard protocol. Pictures were either taken with a Leica DFC 420C camera or with an Aperio ScanScope XT. The border of the tissue area affected by the disease is indicated with a black line in the HE staining (A, D, G, J, M). For patients one and 2 cross sections through the entire lesions are shown (A, D), lesions of patients 3 to 5 (G, J, M) were too large to be entirely processed and therefore only half of the lesion is shown with the necrotic lesion core in the upper right corner. ZN staining (B, E, H, K, N) revealed that AFB were exclusively found in the necrotic areas (red stars). Predominantly clusters of extracellular bacteria were present (C, I, O), however close to the infiltration belt extra- as well as intracellular bacteria (F, L) were observed. Similar findings were made for all 12 lesions analyzed.
Figure 2.
Figure 2.
In depth analysis of the skin tissue in the necrotic core, the infiltrated area and the healthy tissue area (patient 5). Sections were either stained with Haematoxylin-Eosin (HE; A, F-Q) or Ziehl-Neelsen/Methyleneblue (ZN-M; B-E) according to the WHO standard protocol. Pictures were either taken with a Leica DFC 420C camera or with an Aperio ScanScope XT. A: Overview of the histopathological section from patient 5, depicting the 3 regions (healthy tissue, infiltration, necrotic core) which were found in all early ulcerative lesions. Healthy tissue (F-I) presented with a normal epidermal layer (F), no sub- epidermal infiltration (G), healthy fat cells (H) and intact collagen fibers (I). The infiltrated area (J-M), separating the healthy tissue from the necrotic core, reflected a transition state with a slight thickening of the epidermal layer (J), some sub-epidermal infiltration (K), fat cells which started to round up and shrink (L) and the accumulation of large numbers of infiltrating cells (M). The necrotic core (N-O) presented with strongly elongated rete ridges and an epidermal layer which was more than 3 times thicker than the healthy epidermis (N), a strong sub-epidermal infiltration which was largest above the necrotic core (O), fat cell ghosts which displayed signs of cell death (rounding up, shrinkage, absence of nuclei) (P) and necrotic connective tissue without collagen fibers (Q). In the necrotic core focally clustered AFB (D, E, red stars in A) as well as a secondary infection (B, C; at the open ulcer surface (black box A)) could be observed.
Figure 3.
Figure 3.
TUNEL staining. The TUNEL protocol allows the staining of low molecular weight DNA fragments typically occurring during apoptosis (brown staining). Sections of patients one to 5 were stained with the “In Situ Cell Death Detection Kit, POD” (Cat. No. 11684817910) from Roche according to the manufacturer's protocol (protocol used was for tissue sections with high unspecific background). Counter stain was done with Haematoxylin. Pictures were either taken with a Leica DFC 420C camera or with an Aperio ScanScope XT. Overview over tissue sections reveals a diffuse staining of the necrotic lesion core (A1-A4, B1) and no staining of the healthy tissue area. Increased magnification (B2, B3) showed a distinct staining of single cells in the belt of infiltrating cells (B1 green star, B2) representing cells currently undergoing apoptosis and a more dispersed staining inside the necrotic core (B1 red star, B3) representing leftover DNA fragments from the initial infiltration. Similar findings were made for all 12 lesions analyzed.
Figure 4.
Figure 4.
Composition of the immune infiltrate which surrounds the necrotic core. Serial sections from the lesion of patient 5 were stained by immunohistochemistry. The following antibodies were used according to the manufacturer's protocol: Elastase (polymorphonuclear neutrophils, NP57, Dako, M0752), CD3 (T lymphocytes, Dako, A0452), CD68 (macrophages/monocytes, KP1, Dako, M0814), and CD20 (B lymphocytes, 7D1, Novocastra, NCL-CD20–7D1). Pictures were either taken with a Leica DFC 420C camera or with an Aperio ScanScope XT. CD20 positive B-cells (A1, B1) were present in clusters while CD3 positive T-cells (A2, B2), N-elastase positive Neutrophils (A3, B3) and CD68 positive macrophages (A4, B4) were present throughout the infiltration belt in a layered manner. Accumulations of neutrophils were additionally present close to the secondary infection (A3, red dotted circle).
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References

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