Hepatic Topology of Glycosphingolipids in Schistosoma mansoni-Infected Hamsters

Abstract

Schistosomiasis is a neglected tropical disease caused by worm parasites of the genus Schistosoma. Upon infection, parasite eggs can lodge inside of host organs like the liver. This leads to granuloma formation, which is the main cause of the pathology of schistosomiasis. To better understand the different levels of host-pathogen interaction and pathology, our study focused on the characterization of glycosphingolipids (GSLs). For this purpose, GSLs in livers of infected and noninfected hamsters were studied by combining high-spatial-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) with nanoscale hydrophilic interaction liquid chromatography tandem mass spectrometry (nano-HILIC MS/MS). Nano-HILIC MS/MS revealed 60 GSL species with a distinct saccharide and ceramide composition. AP-SMALDI MSI measurements were conducted in positive- and negative-ion mode for the visualization of neutral and acidic GSLs. Based on nano-HILIC MS/MS results, we discovered no downregulated but 50 significantly upregulated GSLs in liver samples of infected hamsters. AP-SMALDI MSI showed that 44 of these GSL species were associated with the granulomas in the liver tissue. Our findings suggest an important role of GSLs during granuloma formation.

Figure 1

Nano-HILIC MS/MS analysis for GSL profiling. (a) Tandem mass spectrum for a singly charged precursor ion at m/z 1430, assigned as HexNac₂Hex₃Cer 18:1;O2/16:0, based on headgroup and backbone fragment ions. (b) Principal component analysis of nano-HILIC MS/MS data in positive-ion mode with “●” for bs-infected, “◊” for ss-infected, “□” for noninfected hamster, and “+” for quality control samples. (c) Extracted ion chromatogram (EIC) for GSLs from the liver of bs-infected hamsters. (d) Histograms for GSL species based on nano-HILIC MS/MS data. Black lines above two bars indicate the difference between the two corresponding samples, with “***” representing a significant difference with p < 0.001 and “**” with p < 0.01, respectively. Error bars show the standard error.

Figure 2

AP-SMALDI analysis of neutral GSLs. (a) Microscopic image of an S. mansoni-liver tissue section of bs-infected hamster, with yellow arrows exemplarily pointing at S. mansoni eggs and orange-dotted circles highlighting granulomas. (b) RGB image corresponding to the microscopic image in (a), showing Fuc₃HexNac₆HexCer 20:0;O3/16:0 ([M + K]⁺, at m/z 2442.2211) in red, HexNac₂Hex₃Cer 18:1;O2/16:0 ([M + K]⁺, at m/z 1468.7919) in green, and HexNacHex₃Cer 18:1;O2/16:0 ([M + K]⁺, at m/z 1265.7134) in blue. Magnifications of parts (a,b) are shown in parts (e,f). (c) Ion image of a ss-infected hamster liver tissue section showing m/z 1468.7939 with the corresponding microscopic image (g). (d) Ion image of a noninfected hamster showing m/z 1468.7946 with the corresponding microscopic image (h). All scale bars are 250 μm. (i) Semiquantitative evaluation of ion images of Fuc₃HexNac₆HexCer 20:0;O3/16:0, HexNac₂Hex₃Cer 18:1;O2/16:0, and HexNacHex₃Cer 18:1;O2/16:0, with a 50 × 50 pixel ROI showing the intensity per pixel for n = 3 with standard error as error bars. Red—bs-infected sample ROI with granuloma included, pink—bs-infected samples without granuloma included, green—ss-infected sample, and blue—noninfected sample. Black lines centered above two bars indicate the difference between the two corresponding ROIs, with “***” representing a significant difference with p < 0.001, “**” with p < 0.01, and “*” with p < 0.05. “n.s.” indicates a nonsignificant difference. Error bars show the standard error.

Figure 3

AP-SMALDI analysis of acidic GSLs. (a) Microscopic image of a liver tissue section of a bs-infected hamster, with yellow arrows exemplarily pointing at S. mansoni eggs and orange-dotted circles highlighting granuloma. (b) RGB image corresponding to the microscopic image in (a), showing NeuAcHex₂Cer 18:1;O2/16:0 ([M–H]⁻ at m/z 1151.7058) in red, NeuGcHex₂Cer 18:1;O2/16:0 ([M–H]⁻ at m/z 1167.7008) in green, and SHexCer 18:1;O2/16:0 ([M–H]⁻ at m/z 778.5148) in blue. Magnifications of (a,b) are shown in (c,f). (d) Ion image of a liver tissue section of a noninfected hamster of NeuGcHex₂Cer 18:1;O2/16:0 ([M – H]⁻ at m/z 1167.6977) with the corresponding microscopic image (e). (g) Ion image of a liver tissue section of an ss-infected hamster of NeuGcHex₂Cer 18:1;O2/16:0 ([M – H]⁻ at m/z 1167.6987) with corresponding the microscopic image (h). Scale bars indicate a length of 250 μm. (i) Histograms for the GSL species shown in the RGB-overlay based on the semiquantitative analysis of AP-SMALDI data. Black lines centered above two bars indicate the difference between the two corresponding ROIs, with “***” representing a significant difference with p < 0.001, “**” with p < 0.01, and “*” with p < 0.05. Error bars show the standard error.

Figure 4

Increasing the lateral resolution enables the localization of substructures in S. mansoni eggs. (a) RGB overlay images of three granulomas measured with a 15 μm step size, (b) 10 μm step size, and (c) 3 μm step size using an experimental AP-SMALDI imaging setup, showing HexCer 20:0;O3/16:0 ([M + K]⁺ at m/z 784.5715) in red, HexNac₂Hex₃Cer 18:1;O2/16:0 ([M + K]⁺, at m/z 1468.7913) in green, and PC 38:1 ([M + K]⁺ at m/z 854.6042) in blue.