Table 1.
Baseline characteristics of the study population and levels of cholesterol, ApoA-I and PON1.
Fig 1.
Multibacillary leprosy patients display lower levels of HDL-cholesterol.
Box-plots represent total cholesterol (A) levels from HC (n = 12), Mb pre-MDT (n = 8), and Pb pre-MDT (n = 6). (B) HDL-cholesterol levels of HC (n = 12), Mb pre-MDT (n = 8), and Pb pre-MDT (n = 6). (C) HDL-cholesterol levels of Mb pre-MDT (n = 8) and Mb post-MDT (n = 6) (B). (D) HDL-cholesterol levels of Pb pre-MDT (n = 6) and Pb post-MDT (n = 7). Median and min-max values are indicated by lines. (A) and (B) HC, Mb pre-MDT and Pb-MDT comparisons were evaluated by Kruskal-Wallis non-parametric and Dunn’s tests. (C) Mb and (D) Pb patients were evaluated with Mann-Whitney non-parametric test. ***p≤ 0.0001.
Fig 2.
Isolation of enriched HDL fractions.
(A) Plasma protein distribution on HPLC gel filtration chromatography fractions (black line, left axis, mAU, milli-absorbance unit, detected at 280 nm) and total cholesterol levels of fractions (red line, right axis, mg/dL), determined by enzymatic assay. (B) 7.5% native- and (C) 15% SDS-PAGE, and (D) ApoA-I immunoblotting profile of HPLC fractions (68–84). M: standard protein markers.
Fig 3.
The lipidomic profile revealed altered composition of HDL particles of multibacillary leprosy patients before treatment.
Raw UPLC-MS data collected in positive ionization mode were processed and analyzed by XCMS, followed by normalization through the quantile method. Subsequently, the molecular features (MFs) were grouped into spectra through RAMClustR. In this approach, each spectrum represents a “compound” with adducts, isotopes and monoisotopic mass. The intensities of 1260 “compounds” were used to perform a principal component analysis. HC (n = 6, black dots), Pb pre-MDT (n = 5, grey dots), Mb pre-MDT n = 4, blue dots).
Fig 4.
HDL from multibacillary leprosy patients displays altered functions.
(A) Inhibition of ROS (reactive oxygen species) by HDL purified from leprosy patients and HC were measured by flow cytometry analysis. Data are expressed by the median fluorescence intensity (MFI). HC (n = 7), Mb pre-MDT (n = 5) and Pb pre-MDT (n = 5) (B) Expression of monocyte chemoattractant protein-1 (MCP-1) modulated by HDL from leprosy patients and HC in HCAEC cells. HC (n = 7), Mb pre-MDT (n = 5), and Pb pre-MDT (n = 6) (C) Interleukin-6 (IL-6) response to HDL of HC and leprosy patients expressed relative to response of HCAEC cells stimulated with LPS (0.5 μg/mL). HC (n = 5), Mb pre-MDT (n = 7), Mb post-MDT (n = 5). (D) Pb, Mb and HC HDL-cholesterol efflux from THP-1 differentiated macrophages. HC (n = 7), Mb pre-MDT (n = 5), and Pb pre-MDT (n = 4). Median and min-max values are indicated by lines. Group comparisons were evaluated using Kruskal-Wallis non-parametric and Dunn’s test. *p≤0.05 and **p≤ 0.001.
Fig 5.
Plasma levels of ApoA-I are drastically reduced in multibacillary leprosy patients.
Box-plots represent levels of ApoA-I in the plasma (A) of HC (n = 8), Mb pre-MDT (n = 5), and Pb pre-MDT (n = 6). (B) ApoA-I in the plasma of Mb pre-MDT (n = 5), Mb post-MDT (n = 6), Pb pre-MDT (n = 6), and Pb post-MDT (n = 6). (C) Plasma PON1 levels from HC (n = 7), Mb pre-MDT (n = 6), and Pb pre-MDT (n = 5). determined by EIA. Median and min-max values are indicated by lines. (A) and (C) HC, Mb pre-MDT and Pb-MDT comparisons were evaluated with Kruskal–Wallis nonparametric and Dunn’s tests. (B) Mb and Pb patients were evaluated with the Mann Whitney non-parametric test. **p≤ 0.001, ***p≤ 0.0001. The absence of p-value indicates non-significant differences.
Fig 6.
M. leprae was detected in the liver of a multibacillary patient and can decrease ApoA-I expression in HepG2 cells.
(A, B, C) Photomicrography of a section from a Mb patient liver autopsy. (A) H&E-stained hepatic parenchyma with vascular congestion (stars) steatosis (black arrows). Extended portal spaces (arrowheads). Inflammatory infiltrate (white arrows); Magnification: 400x, inset 1000x. Fite-Faraco stained Virchow’s cells with granular AFB (stars) in the inflammatory infiltrate (B) and in hepatic sinusoids (C); Magnification: 1000x, inset 400x. (D) Immunofluorescence image of HepG2 cells infected with M. leprae labeled in green (PKH) and nuclei in blue with DAPI; Scale bar, 5 μm. (E) ApoA-I immunoblotting from HepG2 treated with live M. leprae in different MOIs (1:1, 10:1, 25:1 and 50:1). (-) = Unstimulated. A representative experiment of four separate experiments is shown.