Hepatic Topology of Glycosphingolipids in Schistosoma mansoni-Infected Hamsters

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.


Supplementary Protocol 1: Sample preparation for nano-HILIC MS/MS measurements
For nano-HILIC MS/MS, GSLs were extracted as follows.Starting with hamster liver homogenate, 240 µL water, 640 µL methanol and 320 µL chloroform were added, followed by a two-hour incubation at 38 °C with intermediate mixing every 15 minutes.The suspension was centrifuged with 1400 g for 5 minutes.The supernatant was removed and kept on ice.
With the remaining pellet, the previous steps were repeated and the supernatants were combined.Solvents were evaporated under a stream of nitrogen.Subsequently, the samples were dissolved in 1.5 mL of 0.1 mol/L sodium hydroxide in methanol and incubated at 38 °C for saponification of phospholipids.This was followed by neutralization of the sample solutions with glacial acetic acid and evaporation of the solvents under a nitrogen flow.

Figure S3 :
Figure S3: (a) Histograms for endogenous SM compounds showing the peak areas.Error bars indicate the standard deviation.(b) Representative ion-image of SM 40:1;O2 of a liver tissue section of a bs-infected hamster, indicating an almost homogenous distribution across the section with no signal for the eggs.(c) PCA plot including all three biological replicates with three technical replicates each for non-infected (blue), ss-infected (green) and bs-infected (red) GSL extracts based on nano-HILIC MS/MS data.The data point for bs77_1, highlighted by an orange circle, was not included in further statistical evaluation.

Figure S4 :
Figure S4: Box plot showing the coefficient of variation (CV).The %CV was calculated for each identified GSL species in positive-as well as negative-ion mode for the pooled QC sample, which was measured as a replicate five-times for each polarity.l

Figure S8 :
Figure S8: (a) Granuloma model of the pre-granulomatous exudative (PE) stage (a) with B and T cells surrounding the S. mansoni eggs.(b) Granuloma model of the exudative-productive (EP) stage, representing a highly ordered structure with eosinophils and macrophages as the inner layer around the egg, surrounded by collagen fiber and hepatic stellate cells as a middle layer and with B and T cells as the outer layer.In both granuloma stages, cell types with low abundance are not included in the model.

Supplementary Protocol 2: H&E staining protocol Hematoxylin
For purification and desalting, dried GSLs extracts were dissolved in chloroform/methanol/water (3:98:74, v/v/v) and applied to a C18-SPE cartridge (Chromabond C18ec, Macherey and Nagel, Düren, Germany), according to Wuhrer et al.1Before applying the sample, the cartridges were washed with 10 column volumes of methanol, 10 column volumes and eosin (H&E) staining was performed after AP-SMALDI MSI measurements.First, matrix was rinsed off with ethanol and then dehydrated for 2 min in ethanol followed by a 2 min incubation in 70% ethanol.Then the sample was incubated for 2 min in 40% ethanol followed by a 2 min incubation step in HPLC-grade water.Staining with hematoxylin for 12 min, washing with tap water for 15 min and staining with 1% Eosin Y solution for 1 min and differentiation with HPLC-grade water for 2 min, 40% ethanol for 2 min, 70% ethanol for 2 min and 100% ethanol for 2 min were subsequently carried out.Clearing with xylol for 2 min and covering with Eukitt and a cover slide completed the staining protocol.Supplementary

Table 1 .
Protocols for matrix application, sublimation parameters for DHAP and pneumatic spraying parameters for used matrices