Table 1.
Characterization of skin biopsies and clinical data of PB/ MB leprosy patients and controls.
Table 2.
Characterization of skin biopsies and clinical data of RR leprosy patients.
Table 3.
Characterization of skin biopsies and clinical data of ENL leprosy patients
Table 4.
Antibody, source, dilution.
Fig 1.
MAC and C3d deposition in skin of paucibacillary and multibacillary leprosy patients.
Representative immunohistochemical stainings of skin sections from control (A and B), paucibacillary (C and D) and multibacillary (E and F) for C3d, detecting C3d, (C and E) or C9, detecting MAC (D and F). (magnification; 50 μm) showing immunoreactivity for C3d within the dermis layer of the skin of both paucibacillary (C) and multibacillary (E) (in brown) (see arrow). In addition, a strong MAC immunoreactivity was found within the dermis layer of the skin of multibacillary patients (F) (see arrow), indicating abundant local deposition of the active terminal complement product MAC in lesions of multibacillary patients. The control biopsies of skin and nerve are negative for C3d and C9 (A, B). Quantification of the staining (G and H), shows a significant higher amounts of C3d and MAC deposits in skin lesions of multibacillary compared to paucibacillary patients (p = <0.05 and p = <0.001 respectively). Error bars indicate standard error of the mean.
Fig 2.
Representative stainings of skin sections from paucibacillary patients for H&E (A, B Zoom) and immunofluorescence for CD3+ T cells and C3d (C) CD68+ macrophages and C3d (D) CD21 detecting the CR2 receptor and C3d (E) or CD20+ B cells and C3d (F). The H&E staining shows abnormal granulomatous lesions in the skin (A). A zoom in of the H&E staining shows granulomas with epithelioid cells surrounded by lymphocytes (B) Immunofluorescence on the sections indicated that the T cells, the CR2 receptor and B-cells all co-localized with C3d in skin lesions of paucibacullary patients.
Fig 3.
H&E staining of LL leprosy skin (magnification; 100 μm) (A) and zoom-in (magnification; 25 μm) showing giant epithelioid cells in the skin lesion. Double staining of LL leprosy skin for macrophage marker CD68 (red) with C3d (green) (C) and MAC (green) (D) showed co-localization of both complement markers with macrophages (magnification; 25 μm). Staining for MAC with the marker SMI31 that visualizes the nerves or LAM showed that these markers co-localized indicating that MAC attacks the axons in the skin and that LAM could be a trigger for the complement activation in the lesions.
Fig 4.
Representative immunohistochemical stainings of skin sections from BL (A and B), ENL (C and D) and RR (E and F) for C3d (A, C and E) and MAC (B, D and F) (magnification; 100 μm). Quantification of the staining (G and H), shows a significant higher amounts of C3d and MAC deposits in skin lesions of BL compared to RR patients (p = <0.05 and p = <0.01 respectively). In addition, patients that developed ENL had a higher amount of MAC deposition in the skin compared to BL patients without a reaction (p = <0.01). Error bars indicate standard error of the mean.
Fig 5.
Representative immunohistochemical stainings of skin sections from Control (A), paucibacillary (B) and multibacillary (C) patients for LAM (magnification; 100 μm). Quantification of the staining (D), shows a significant higher amounts of LAM deposits in skin lesions of multibacillary compared to paucibacillary patients (p = <0.001). Error bars indicate standard error of the mean.
Fig 6.
Representative immunohistochemical stainings of skin sections from BL (A), ENL (B) and RR(C) patients for LAM (magnification; 100 μm). Quantification of the staining (D), shows a significant higher amounts of LAM deposits in skin lesions of RR and ENL compared to BL patients without a reaction (p = <0.05 and p = <0.05, respectively). No statistical difference was found between ENL and RR patients in the percentage of LAM deposition in skin lesions. Error bars indicate standard error of the mean.
Fig 7.
Bacterial Index (BI) and LAM deposition are associated with C3d and MAC deposition in skin lesions of leprosy patients.
The amount of C3d (a,c) and C9 (b,d) immunoreactivity significantly correlated with the BI and LAM deposition in skin of paucibacillary and multibacillary (Pearson’s correlation for BI, r = 0.99909, p = <0.0001 and r = 0.9612, p = <0.0001 respectively) (Pearson’s correlation for LAM, r = 0.9578, p = <0.0001 and r = 0,8585, p = <0.0001 respectively), indicating an association between the M.leprae BI or LAM and complement activation in leprosy skin.
Fig 8.
Representative staining pattern with an antibody against for LAM, detecting M.leprae, or C9, detecting MAC, in skin biopsies of a treated leprosy patients showing M.leprae antigen LAM (A) as well as MAC (B) persist in the skin after completion of treatment (magnification; 50 μm).