Fig 1.
Immunological analysis of patients with PKDL.
Schematic analyses performed on patient groups and healthy controls.
Table 1.
Study Population.
Fig 2.
Decreased expression of TLR-2 and -4 and altered redox status within monocytes.
A-B: Representative data showing expression of TLR-2 (A) and TLR-4 (B), within CD14+ monocytes in a healthy control, a patient with PKDL (Pre t/t) and post treatment (Post t/t). Isotype control staining is also shown. *Scatter plots showing frequency of CD14+ monocytes expressing TLR-2 or TLR-4 in healthy controls (●), patients with PKDL (Pre t/t, ■) and post treatment (Post t/t, ▲). *The proportion of CD14+TLR-2+ and CD14+TLR-4+ monocytes was calculated by dividing the percentages of upper right quadrant with the sum of upper and lower right quadrant. C: Scatter plots showing intracellular NO in circulating monocytes of healthy controls (●), patients with PKDL (Pre t/t, ■) and post treatment (Post t/t, ▲). Representative histogram overlay showing DAF-2T fluorescence in monocytes from a healthy control (—), patient with PKDL (…) and on completion of treatment (—-). D: Scatter plots of intramonocytic generation of ROS as for C above. A representative histogram overlay showing CM-DCF fluorescence, as for D above. E: Scatter plots indicating levels of intracellular non-protein thiols as for C above. A representative histogram overlay showing CMF fluorescence as for D above. For analysis, monocytes were initially gated on their forward vs. side scatter characteristics followed by fluorescence of CD14.
Fig 3.
Raised expression of PPAR-γ was accompanied by an increased expression of arginase-1 and mannose receptor in circulating monocytes.
A-C: Representative mRNA expression in circulating monocytes of PPAR-γ (A), arginase-1 (B) and mannose receptor (C) sourced from healthy controls (N 1–3), patients with PKDL (Pre 1–3) and post treatment (Post 1–3). Scatter plots indicate RT-PCR products in healthy controls (●), patients with PKDL (Pre t/t, ■) and after treatment (Post t/t, ▲). These RT-PCR products were quantified by densitometric analysis after normalization with β-actin, along with before and after plots of the same. D: Representative quadrant plot showing frequency of CD206 in a healthy control, a patient with PKDL (Pre t/t) and post treatment (Post t/t). Scatter plots shows frequency of CD206 within CD14 gated monocytes of healthy controls (●), patients with PKDL (Pre t/t, ■) and after treatment (Post t/t, ▲). The proportion of CD14+CD206+ monocytes was calculated by dividing the percentages of upper right quadrant with the sum of upper and lower right quadrant.
Fig 4.
Analysis of intramonocytic status of cytokines in PKDL.
Scatter plots indicating the % of CD14+ monocytes expressing the cytokine in healthy controls (●), patients with PKDL (Pre t/t, ■) and after treatment (Post t/t, ▲). The proportion of CD14+IL-6+, CD14+IL-1β +, CD14+IL-8+, CD14+IL-12p40+, CD14+LAP-TGF-β1+monocytes was calculated by dividing percentages of the upper right quadrant with sum of the upper and lower right quadrant (as shown in S2 Fig).
Fig 5.
Lesional macrophages showed a raised expression of PPAR-γ, arginase-1 and mannose receptor.
A and B: Representative mRNA expression profile of PPARG (A) and ARG1 (B) in skin samples from healthy controls (N 1–3), patients with PKDL pre (Pre 1–3) and post treatment (Post 1–3). Scatter plots indicate RT-PCR products in healthy controls (●), patients with PKDL (Pre t/t, ■) and after treatment (Post t/t, ▲). These RT-PCR products were quantified by densitometric analysis after normalization with β-actin, along with before and after plots of the same. C: Expression of arginase-1 (green, Panels 1 and 2) in CD68+ macrophages (red, Panels 1 and 2) at the lesional site of a patient with PKDL (Pre t/t) and post treatment (Post t/t). Nuclei are shown in blue (DAPI, Panels 1 and 2). Co-localization of macrophage and arginase-1 appears as yellow. Figures were captured in 400X magnification. D: Representative mRNA expression profile of CD206 in dermal lesions of healthy controls (N 1–3), patients with PKDL pre (Pre 1–3) and post (Post 1–3) treatment. Scatter plots are as in A-B, above. E: Expression of mannose receptor (CD206, green, Panels 1 and 2) in CD68+ macrophages (red, Panels 1 and 2) at the lesional site of a patient with PKDL (Pre t/t) and post treatment (Post t/t). Nuclei are shown in blue (DAPI, Panels 1 and 2). Co-localization of CD68+ macrophages and mannose receptor (CD206) appears as yellow.
Fig 6.
Status of Vitamin D3 and associated downstream signalling molecules in PKDL.
A: Plasma levels of hydroxylated vitamin D3 in healthy controls (●) and patients with PKDL pre (Pre t/t, ■) and post treatment (Post t/t, ▲) along with before and after plots of the same. B: Representative mRNA expression profile of CYP27B1, VDR, LL-37 mRNA in circulating monocytes of healthy controls (N1-3), patients with PKDL (Pre1-3) and post treatment (Post1-3). Scatter plots indicate RT-PCR products in healthy controls (●), patients with PKDL (Pre t/t, ■) and after treatment (Post t/t, ▲). These RT-PCR products were quantified by densitometric analysis after normalization with β-actin, along with before and after plots of the same. C: Representative mRNA expression of CYP27B1, VDR, LL-37 in skin samples from healthy controls (N 1–3), patients with PKDL (Pre 1–3) and post treatment (Post 1–3). Scatter plot indicates expression as in B above.