Analytical and Bioanalytical Chemistry (2022) 414:3653–3665
https://doi.org/10.1007/s00216-022-04006-6
RESEARCH PAPER
Changes in the lipid profile of hamster liver after Schistosoma mansoni 
infection, characterized by mass spectrometry imaging and LC–MS/MS 
analysis
Katja R. Wiedemann1 · Alejandra Peter Ventura1 · Stefanie Gerbig1 · Martin Roderfeld2 · Thomas Quack3 · 
Christoph G. Grevelding3 · Elke Roeb2 · Bernhard Spengler1
Received: 14 October 2021 / Revised: 18 January 2022 / Accepted: 3 March 2022 / Published online: 23 March 2022
© The Author(s) 2022
Abstract
Schistosomiasis, caused by the human parasite Schistosoma mansoni, is one of the WHO-listed neglected tropical diseases 
(NTDs), and it has severe impact on morbidity and mortality, especially in Africa. Not only the adult worms but also their 
eggs are responsible for health problems. Up to 50% of the eggs produced by the female worms are not excreted with the 
feces but are trapped in the host tissue, such as the liver, where they provoke immune responses and a change in the lipid 
profile. We built up a database with 372 infection markers found in livers of S. mansoni-infected hamsters, using LC–MS/
MS for identification, followed by statistical analysis. Most of them belong to the lipid classes of phosphatidylcholines 
(PCs), phosphatidylethanolamines (PEs), and triglycerides (TGs). We assigned some of these markers to specific anatomical 
structures by applying high-resolution MALDI MSI to cryosections of hamster liver and generating ion images based on the 
marker list from the LC–MS/MS experiments. Furthermore, enrichment and depletion of several markers were visualized.
Keywords Schistosomiasis · AP-SMALDI · Mass spectrometry imaging · Infection · Host-parasite interaction · 
Granuloma · Parasites · Schistosoma mansoni
Abbreviations LDL L ow-density lipoprotein
AA, 20:4 A rachidonic acid LPE L ysophosphatidylethanolamine
CL C ardiolipin MALDI MSI M atrix-assisted laser desorption/ionization 
CE  Cholesterol ester mass spectrometry imaging
DAN  1,5-Diaminonaphthalene MMPE M onomethylphosphatidylethanolamine
DG  Diglyceride NTDs N eglected tropical diseases
DHB  2,5-Dihydroxybenzoic acid PC  Phosphatidylcholine
DMPE D imethylphosphatidylethanolamine PE P hosphatidylethanolamine
DHA, 22:6 D ocosahexaennoic acid PS P hosphatidylserine
ECN E quivalent carbon number PZQ P raziquantel
FDR F alse discovery rate PCA P rincipal component analysis
LC–MS/MS L iquid chromatography tandem mass So  Sphingosine
spectrometry TG  Triglyceride
*  Bernhard Spengler Introduction
 bernhard.spengler@anorg.chemie.uni-giessen.de
1 Institute of Inorganic and Analytical Chemistry, Justus Infection with the blood fluke Schistosoma mansoni leads to 
Liebig University Giessen, Giessen, Germany severe health issues. The resulting disease schistosomiasis 
2 Gastroenterology, Justus Liebig University Giessen, Giessen, can occur in two different stages, an acute and a chronic 
Germany form. During acute infection, patients typically suffer from 
3 Institute for Parasitology, Justus Liebig University Giessen, non-specific symptoms such as fever, fatigue, malaise, 
Giessen, Germany and cough. When the worms start to migrate, abdominal 
Vol.:(012 3456789)
3 654 Wiedemann K. R. et al.
symptoms can occur [1]. Main symptoms of chronic infec- they appear to be important for the survival of the host by 
tion are abdominal pain, diarrhea or bloody stool, and protecting hepatocytes from toxins released by the eggs. 
inflammation of inner organs such as the liver, spleen, and In addition, granuloma results from inflammatory pro-
gut [2]. cesses that finally lead to liver fibrosis [7]. Granuloma 
Humans infected with S. mansoni excrete parasite eggs in general mainly consists of macrophages which mass 
with their feces. When the eggs enter fresh water, the first together [9]. However, liver granulomas are more hetero-
larval stage, miracidia hatch and infect their intermediate genic in cell types and contain also T and B lymphocytes 
host, a snail of the genus Biomphalaria. Inside the snails, as well as eosinophils and mast cells [10]. Furthermore, 
the parasite multiplies asexually and develops after 4 to it appears that granuloma formation differs in naturally 
6 weeks to male and female cercariae, which are then infected hosts from those observed under laboratory 
released into water. Cercariae penetrate the skin of their conditions [11]. It is known that S. mansoni infection in 
vertebrate host and migrate to the blood vessels. During general leads to lower cholesterol, low-density lipopro-
this phase, cercariae transform into schistosomula and sub- tein (LDL), and TG levels in host blood streams [12, 13]. 
sequently to adult schistosomes. In the portal vein of the Another study revealed that lipid uptake in hepatic stel-
liver, male and female worms mate and migrate as couples late cells was provoked around trapped eggs [14]. Stanley 
to the mesenteric veins of the gut, where they can live for et al. showed that lower cholesterol blood levels were 
up to 30 years. A unique feature of schistosome biology is caused by factors secreted by the schistosome eggs. Addi-
that the sexual maturation of the female is only achieved tionally, they observed agglomeration of cells with higher 
upon a constant pairing contact with the male. Following lipid contents in the outer parts of the granuloma [15]. 
pairing, the female reproductive organs fully differentiate; The described processes in livers of infected animals and 
the female starts the production of several hundred eggs humans underline the importance of research regarding 
per day, which reach the gut lumen and are excreted by infection-induced metabolic changes.
the host [1, 2]. Investigating lipids has become a highly active research 
Schistosomiasis is classified as an endemic disease area in the last years including instrumental advances, the 
being prevalent in tropical and subtropical regions world- detection of lipid biomarkers for various conditions, unrave-
wide. According to the WHO, schistosomiasis belongs to ling of double-bond positions in fatty acids, and the char-
the NTDs and is mainly spread in Africa, Asia, the Middle acterization of the lipidome of biological samples [16–19]. 
East, the Caribbean, and parts of South America [2, 3]. Although literature is available on the lipid composition 
Improving both hygienic conditions as well as devel- of trematodes, the interaction of parasite and host has not 
oping chemotherapeutics plays an important role in com- been studied on the lipid level yet [20, 21]. Mostly, enzymes 
bating schistosomiasis and preventing the further spread related to lipid metabolism have been investigated to specu-
of the disease [1]. Since 1977, praziquantel (PZQ) is the late about their impact on lipid composition of worms and 
main drug against schistosomiasis [4]. PZQ is effectively the host [22].
targeting all schistosome species, but due to the risk of The parallel use of matrix-assisted laser desorption/ioni-
upcoming resistances against PZQ, new drugs are urgently zation mass spectrometry imaging (MALDI MSI) and liquid 
needed. Additionally, PZQ affects adult but not juvenile chromatography tandem mass spectrometry (LC–MS/MS) 
worms [5]. is a widely used technique to investigate lipidomic profiles 
In case of S. mansoni, about 50% of the eggs are of samples while keeping the lateral information of the ana-
excreted with the feces, the other 50% are mostly trapped lytes. Commonly, lateral resolutions of about 10 µm are used 
in the liver and intestine of the host and secrete anti- [23]. This setup has already been applied to the analysis of 
gens and other factors, which can affect the host [6, 7]. S. mansoni worms [20, 24] and has now been optimized for 
Trapped eggs are the primary reason for the pathologi- egg-infected hamster liver tissue.
cal consequences of schistosomiasis. While the host’s The combination of MALDI MSI and LC–MS/MS is 
immune response is mainly directed towards antigens the key to get a deeper understanding of the interactions 
produced by the schistosome worms during the first between trapped eggs and the surrounding tissue. Analyzing 
weeks of infection, the immune response shifts after 5 infected tissue that still contains the parasites is beneficial to 
to 6 weeks. After pairing, female schistosomes start to get an overview of the metabolic changes in both, the host 
produce eggs, which then provoke the type 2 response of tissue and the eggs. Due to the small size of the S. mansoni 
the host’s immune system [7, 8]. This immune response eggs of about 60–200 µm [25], a high lateral resolution 
is essential for the egg’s movement through the intestinal of the applied MSI technique was essential. To the best 
wall to reach the gut lumen [7]. Due to the reaction of of our knowledge, this is the first study of interactions of 
the tissue, granuloma formation occurs around the eggs S. mansoni eggs (parasite) with the host liver tissue using 
within a few days [1]. Granuloma have a dual function as MALDI MSI.
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Changes in the lipid profile of hamster liver after Schistosoma mansoni infection,… 3655
Materials and methods hematoxylin (5 dips), and rinsed with distilled water before 
mounting in aqueous mountant or glycerin jelly.
Chemicals
A list of chemicals and suppliers is found in Table S1 in MALDI MSI sample preparation
the Supplementary Information.
Fresh frozen liver samples were kept at − 80 °C prior to 
sample preparation. Cryosections of 20 µm thickness with-
Tissue samples out any further fixation were prepared at − 25 °C using a 
Cryostat Microm HM 525 (Epredia, MI, USA). After cut-
To keep the S. mansoni life cycle, Biomphalaria glabrata ting, sections were thaw-mounted onto common glass slides. 
snails served as intermediate hosts and Syrian hamsters Microscopic images were taken using a digital microscope 
(Mesocricetus auratus) as final hosts [26]. The hamster (VHX-5000, Keyence, Neu-Isenburg, Germany). Sections 
model has been established because it is more permissive were kept at − 80 °C until measurement.
for schistosome infection compared to the mouse model, For MS imaging analysis, samples were thawed in a 
which requires more animals (reduction principle of the desiccator for 30 min. For positive-ion mode, 2,5-dihy-
3Rs). Both snails and hamsters were bred in-house (Bio- droxybenzoic acid (DHB) and for negative-ion mode, 
medical Research Center Seltersberg, Giessen, Germany). 1,5-diaminonaphthalene (DAN) were applied. 30 mg/mL 
All animal experiments were approved by the Regierung- of DHB was dissolved in acetone/H2O/trifluoroacetic acid 
spraesidium Giessen (V54-19 c 20/15 h 02 GI 18/10 No. (49.95:49.95:0.1, v:v:v). DAN was prepared at a concentra-
A 14/2017) and performed in accordance with the Euro- tion of 3.3 mg/mL in H 2O/methanol (1:9, v:v). Matrix was 
pean Convention for the Protection of Vertebrate Animals applied using an ultrafine pneumatic sprayer (SMALDIPrep, 
used for experimental and other scientific purposes (ETS TransMIT GmbH, Giessen, Germany) as described else-
No. 123; revised Appendix A). For this study, liver sam- where [31]. In brief, 100 µL DHB and 400 µL DAN solu-
ples of female hamsters were used. Three different groups tion were applied with a flow rate of 10 µL/min and 30 µL/
of hamster livers were examined: non-infected hamsters min, respectively. Nitrogen pressure was adjusted to 1 bar.
were used as controls, hamsters infected with only one 
sex of S. mansoni cercariae (monosex-infected), and ham-
sters infected with both sexes of cercariae (bisex-infected) MALDI MSI analysis
[26–28]. Each group consisted of three biological repli-
cates. Bisex infections were carried out according to estab- For MSI analysis, a high-resolution atmospheric-pressure 
lished protocols and were performed for 46 days, while MALDI imaging ion source (AP-SMALDI5 AF, TransMIT 
monosex infections persisted up to 67 days [29] to ensure GmbH), coupled to an orbital trapping mass spectrometer 
a sufficient quality of the worms. This especially applies (Q Exactive HF, Thermo Fisher Scientific (Bremen) GmbH, 
for female schistosomes that need longer to grow and Bremen, Germany), was employed. Instrumental settings are 
develop in hamsters without male partner. Non-infected listed in Table S2.
controls were age-matched with bisex-infected hamsters. Mass accuracy of ± 1 ppm was maintained by internal cal-
Liver samples were shock frosted in liquid nitrogen imme- ibration to a matrix cluster ion (m/z 716.12461 [5 DHB − 4 
diately after perfusion and subsequently stored at − 80 °C.  H2O +  NH +4] ).
Optical images of all measured samples can be found in 
Figure S14.
H&E staining
OilRed staining After MSI measurements, matrix was washed off with etha-
nol. Afterwards, sections were stained the following way:
Neutral lipids were stained as described before [30]. Briefly, At first, sections were rehydrated using 100%, 70%, and 
8- to 10-µm cut-frozen sections were air-dried on glass 40% ethanol and deionized water, 2 min each. Afterwards, 
slides, subsequently 10 min fixed in 10% formalin, briefly samples were kept in hematoxylin solution for 12 min, fol-
washed with running tap water 1–10 min, and afterwards lowed by 10 min in tap water and 5 min in deionized water. 
rinsed for 2 s with 60% iso-propanol before staining with After 1 min in eosin y solution, samples were dehydrated 
freshly prepared Oil Red O working solution (0.2% in 50% using deionized water, 40%, 70%, and 100% ethanol and 
iso-propanol) for 15 min. After staining, slides were rinsed xylene for 2 min each. Finally, samples were covered with 
for 2 s with 60% iso-propanol, lightly stained with alum Eukitt and a cover slip.
1 3
3 656 Wiedemann K. R. et al.
LC–MS/MS sample preparation principal component analysis (PCA), data were not filtered, 
but row-wise normalized to constant sum. Afterwards, 
For LC–MS/MS experiments, lipid extraction was per- data were transformed by log10 normalization. For marker 
formed according to Breitkopf et  al. [32] with minor search, lipids found in bisex-infected samples were com-
changes. In brief, 10 mg liver homogenate, 100 µL PBS pared to monosex-infected and control samples, respectively. 
buffer, and 350 µL methanol were vortexed for 1 min at Therefore, measurements were grouped according to their 
1500 rpm. Afterwards, 1 mL MTBE was added, and the biological sample group (control, monosex, bisex). For nor-
mixture was shaken for 1 h at 20 °C and 1000 rpm. For better malization, all signal intensities, expressed as peak areas, 
phase separation, 300 µL  H2O was added, mixed for 1 min were divided by the total ion count of one sample. Values 
at 1000 rpm, and centrifuged for 8 min at 13,000 rpm. 1 mL were standardized using z-scores. Afterwards, ANOVA tests 
of the organic upper phase was separated and dried under with a permutation-based false discovery rate (FDR) of up 
nitrogen flow. Dried samples were stored at − 80 °C until to 5% were performed. Significant values were filtered and 
measurement. Prior to analysis, samples were reconstituted Tukey’s post hoc test was performed. The results were hier-
with 100 µL acetonitrile/iso-propanol/H2O (65:30:5, v:v:v). archically clustered, leading to a list of significant “infection 
The whole extraction procedure was done additionally with markers”.
100 µL PBS as extraction blank; reconstitution buffer was These lists (for positive- and negative-ion mode) were 
used as blank. Samples obtained from different animals were used to generate MS images with Mirion [37]. Prior to image 
used for LC–MS/MS analysis and for imaging experiments. generation, no normalization or any other data processing 
was performed. Images were assessed manually for interest-
LC–MS/MS analysis ing distributions and overlaid according to their patterns. 
Identifications in the imaging data are only tentative. They 
Separation was performed on a UHPLC system (Ultimate were identified according to their exact mass by compari-
3000 UHPLC, Thermo Fisher Scientific) equipped with a son with the list of markers identified by LC–MS/MS. Due 
reversed-phase 1.8 µm column (100 X 2.1 mm) (ACQUITY to insufficient signal intensities, no MS/MS analyses were 
UPLC HSS T3, Waters GmbH, Eschborn, Germany). Mobile performed during the MSI measurements.
phase A consisted of H 2O/acetonitrile (40:60, v:v), mobile 
phase B of iso-propanol/acetonitrile (90:10, v:v), both with 
10 mM ammonium formate and 0.1 vol-% formic acid, as Results and discussion
previously published [33]. Column gradient parameters for 
reversed-phase liquid chromatography separation are listed Granuloma around eggs in liver tissue of infected 
in Table S3. Flow rate was kept constant at 0.25 mL/min, animals exhibits an accumulation of lipids
injection volume was 10 µL. All samples of one group were 
measured consecutively. After each group, blanks were It is well known that schistosome infection leads to granu-
injected to clean the column. The gradient was used for both loma formation around eggs deposited in tissues such as 
positive- and negative-ion mode. the liver [7]. Granuloma mainly consist of macrophages and 
After separation, tandem mass spectra were recorded other immune cells [9, 10], which are assumed among oth-
using an orbital trapping mass spectrometer (Q Exactive ers to play important roles in providing nutrients in form of 
HF-X, Thermo Fisher Scientific) for all samples. Heated lipids for the eggs [11]. On the other hand, they shield other 
electrospray ionization parameters are listed in Table S4, hepatocytes from injury [38]. Granulomas are readily vis-
MS/MS parameters are listed in Table S5. ible in the microscopic images of prepared liver sections as 
For quality control, pooled samples, containing 10 µL of shown in Fig. 1A. Staining with oil red revealed an accumu-
all individual samples were added at the beginning, the mid- lation of neutral lipids in the granuloma as shown in Fig. 1B.
dle and the end of the run. LC–MS/MS data were analyzed To distinguish between effects of the eggs and systemic 
using LipidMatch Flow 3.1 [34]. To maximize the identifi- effects of worm infection, bisex-infected samples were com-
cation output, all samples were used as targets and ddMS2. pared to monosex-infected samples, which are expected 
Additionally, the equivalent carbon numbers (EQN) were to produce no eggs. In rare cases, however, non-fertilized 
calculated as EQN = CN − 2∙DB, with CN: carbon number egg-like structures appear in hamsters infected with clonal 
of the fatty acids and DB: double bonds. female cercariae (monosex-infected), which can be accom-
panied by other types of host reactions [39]. For our studies, 
Statistical analysis and MS image generation only monosex-infected samples without eggs were included. 
Non-infected samples were defined as controls to differenti-
Statistical analyses were performed using Perseus [35] ate between healthy (no eggs) and infected (eggs) samples. 
and MetaboAnalyst [36] as described elsewhere [20]. For Microscopic images of the three different sample types 
1 3
Changes in the lipid profile of hamster liver after Schistosoma mansoni infection,… 3657
Fig. 1  Microscopic images of 
eggs with granuloma in livers 
of bisex-infected hamsters. 
(A) Light microscopic image 
of unstained tissue. (B) Tissue 
stained with oil red. Lipid accu-
mulation in granuloma is clearly 
visible. Scale bars are 500 µm
used in this study are shown in Figure S1 in the supplemen- found in Figure S3. All lipid identifications are based on 
tary file. While a detailed histological investigation of the head group and fatty acid fragments with one exception that 
involved cell types in the immune response of the hamster is was only confirmed by class. Using MetaboAnalyst, PCA 
not the scope of this study, further information on the topic was performed. As the scores plot in Figure S5 shows, the 
will be found in the literature [von Bülow et al. 2022, in three sample groups were well separated based on LC–MS/
preparation]. Granulomas were formed due to trapped eggs MS data, already suggesting that the lipid profiles vary sig-
in the bisex-infected (C) samples. The S. mansoni eggs are nificantly. Additionally, the PCA shows all three technical 
clearly visible within the granulomatous area in the zoomed- replicates of the pool samples arranged in very close vicin-
in image (D) of the bisex-infected samples. Tissue areas that ity, indicating that the LC measurements were acquired with 
were subsequently subjected to MS imaging experiments stable performance.
were selected based on microscopic images to ensure granu- In parallel, cryosections of the liver samples were pre-
loma inclusion in case of bisex samples. pared and analyzed in positive- as well as in negative-ion 
During further analysis, microscopic images were used to mode with MALDI MSI, as shown in Fig. 2. For some of 
allocate identified markers to infection-specific morphologi- the infection markers, distribution patterns matching with 
cal structures, such as eggs and granuloma. While overlaying optical images were found by annotating MSI data using the 
optical and MS images, marker localization was confirmed. marker list from LC–MS/MS experiments in Mirion. The 
Due to the high thickness of tissue sections used for MALDI three native color channels (red, green, and blue) were used 
(≈ 20 µm), histological investigation is hindered. Moreover, to overlay the three selected MS images (D), showing sig-
tissue sections can only be stained after the MSI experiment, nificantly different spatial distributions for the three mark-
and cellular structures are affected and partly destroyed by ers. Comparison with the microscopic image revealed their 
the laser irradiation. connection to different compartments of the inflammation. 
An exemplary microscopic image of an H&E-stained sec- All identified markers and their predominant localization (if 
tion after MSI analysis can be found in Figure S2. applicable) are listed in an additional excel sheet (see sup-
plemental information, “infection marker list”).
LC–MS/MS analysis detected infection markers The overlap between the detected m/z data sets of 
LC–MS/MS and MALDI MSI was not perfect. Most of the 
In order to identify markers for S. mansoni infection and detected m/z values were found with only one method. This 
to determine their distributions in the tissue, we combined effect is due to the different ionization mechanisms of ESI 
different mass spectrometric techniques. Experiments were and MALDI that favor ionization of different lipid classes as 
performed with three sample groups (non-infected, mono- well as the formation of different adduct species. While the 
sex-infected, and bisex-infected), comprising three biologi- solvent in LC–MS/MS experiments contained ammonium 
cal replicates of each group. An overview of the workflow formate, and predominantly [M +  NH4]+ ions were formed, 
is given in Fig. 2. The top right part shows a piece of ham- for example, for triglycerides, the same lipid species were 
ster liver that was used for analysis. Following the figure detected as alkali metal adducts with MALDI MSI. To 
counterclockwise, parts of liver samples were homogenized enhance the overlap of the two data sets, the m/z values of 
followed by lipid extraction. Applying LC–MS/MS, 372 the identified lipids in LC–MS/MS were re-calculated. In 
significantly occurring markers were found in the extracts case of triglycerides, m/z values for [M + H ]+, [M + N a]+, 
after lipid identification in positive- and negative-ion mode and [M +  K]+ ions were calculated and were used to create 
with LipidMatch Flow and statistical analysis of the data images. An example can be found in Figure S6 and is dis-
with Perseus. An exemplary LC–MS/MS spectrum can be cussed later in the MSI part. Another aspect that has to be 
1 3
3 658 Wiedemann K. R. et al.
Fig. 2  Hamster liver samples 
were either homogenized or 
cryosectioned. After homog-
enization, lipids were extracted, 
and samples were analyzed 
using LC–MS/MS. With the 
help of LipidMatch Flow and 
Perseus, identified signals 
were annotated and markers 
were identified that occurred 
with statistically determined 
significance. After cryosec-
tioning, matrix was applied, 
and samples were measured 
using MALDI MSI. Using 
the software Mirion and the 
marker list from LC–MS/MS 
experiments, MS images were 
generated: (A) m/z 500.275684, 
LPE(20:4), [M − H ]−; (B) 
m/z 866.592639, PS(42:4), 
[M −  H]−; (C) m/z 776.526946, 
MMPE(16:0_22:6), [M −  H]−; 
(D) RGB overlay of MS images 
A, B, C
kept in mind is the difference in sample preparation. While the host organism and the cercarium develops into an adult 
samples for LC were homogenized and lipid extraction was worm that resides in different organs of the host. In a previ-
performed, whole tissue sections were used for MSI experi- ous study, up to 85% of the unpaired female worms and 65% 
ments. Therefore, matrix effects might play an important of male worms were found in mouse liver after 8 weeks. 
role regarding comparability of MSI data to LC–MS/MS The female worm induced an accumulation of inflamma-
data. Matrix effects are present also for LC measurements tory cells in blood vessels and the surrounding liver tissue 
but should be smaller compared to MSI. Furthermore, some [40–42]. These effects also play a role when paired worms 
markers detected in MSI measurements might be absent in from a bisex-infection are present, but morbidity-inducing 
LC data due to low solubility. liver damage is ultimately caused by the deposited eggs. 
More markers distinguish between bisex-infected and Altogether, the hepatic lipid composition of bisex-infection 
control samples than between bisex- and monosex-infected shows more similarities to a monosex-infection than to the 
samples, as seen in Fig. 3. Markers found for monosex- control which might reflect the common changes due to 
infected samples were mostly those also found for bisex- worm presence in both infection states.
infected samples (35 “enriched” markers in Fig. 3). The Changes in lipid composition were mostly found in the 
effects of S. mansoni infection on the lipid composition of groups of PCs, PEs, and TG (Fig. 4). All subgroups of one 
the host liver tissue are manifold. Although infection with major phospholipid class were summed in the same group. 
monosex cercariae does not result in egg deposition and As an example, all PEs, lyso-PEs, oxidized PEs and oxidized 
granuloma formation, the unpaired parasites still provoke lyso-PEs were summed up and shown in the PE bar (for full 
1 3
Changes in the lipid profile of hamster liver after Schistosoma mansoni infection,… 3659
Fig. 3  Venn diagram showing the numbers of infection markers since further biological analysis is needed to verify that the eggs actu-
found. Many more infection markers were found to be enriched when ally take up and metabolize the lipids. Here, the terms are meant to 
comparing bisex-infected with control samples than with monosex- describe changes in signal intensities found in LC–MS/MS measure-
infected samples (A). The same holds for depleted infection markers ments
(B). The terms “enriched” and “depleted” have to be used carefully 
60
50
40
30
20
10
0
enriched in bisex depleted in bisex enriched in bisex depleted in bisex
rel. ct o nctornotlrol rel.c ton ctroonltrol rel.m too nmoosneoxsex rel.m to nmoosneoxsex
PE CE Cer CL DG PC PS TG PI PG PA SM other
Fig. 4  Enrichment and depletion of lipids of several lipid classes in Markers were found by comparing the groups of bisex-infected sam-
bisex-infected hamster liver, relative to non-infected controls or mon- ples to monosex-infected or control samples. No individual compari-
osex-infected hamsters. Significantly more enriched/depleted markers son between the three samples of one sample group was performed. 
were observed relative to controls than to monosex-infected samples. Lipids with a FDR of up to 5% were assumed as infection markers
information on the lipid species detected, please refer to the the sample groups. While no striking difference was found 
“Infection marker” table in the Supplementary information). for PC and PE markers, triglycerides revealed a varying 
Interesting lateral distributions of such subgroups are dis- number of double bonds in the fatty acid chains. The 48 
cussed in more detail below in the MS imaging part. Both TG markers that were found enriched in bisex-infected 
enrichment and depletion were found due to egg deposi- samples compared to control had an average number of 
tion. The terms “enriched” and “depleted” have to be used double bonds of n = 8.4 while the 51 TG markers depleted 
carefully here, since further biological analyses are needed in bisex-infected samples compared to control samples 
to verify that the eggs take up and metabolize the depleted had an average double bond number of n = 3.1. The carbon 
lipids. Until then, the terms are meant to describe changes in number differed only in the range of the standard devia-
signal intensity during LC–MS/MS measurements with no tion. By examining the “Infection marker” table in the Sup-
information about depletion or enrichment location. plementary information, one can find no arachidonic acid 
The majority of markers belong to the TGs, followed by (AA, 20:4) or docosahexaennoic acid (DHA, 22:6) in the 
PEs and PCs. These lipid classes are known for their good markers of the control samples, but one or both of these 
detectability in LC–MS measurements. We calculated the fatty acids appear in 52% of TGs enriched in bisex-infected 
average number of carbon atoms and double bonds in the samples. It was shown previously that AA treatment of 
detected PC, PE, and TG markers for comparison between hamsters infected with S. mansoni reduced worm burden 
1 3
number of lipid markers per class
3 660 Wiedemann K. R. et al.
and the number of eggs [41], so the presence of AA in Lipid distributions revealed by MALDI MSI
bisex-infected samples might be a protective response of 
the immune system. The treatment with DHA also resulted In Fig. 5A and C, lateral distributions of selected markers 
in reduced worm burden and lower egg count, but the effect defined in our LC–MS/MS experiments are shown. For all 
was not as clear as for AA. images, MMPE(16:0_22:6) (monomethylphosphatidyl etha-
Enrichment was found in LC–MS/MS measurements nolamine) was chosen to visualize the liver tissue in blue. 
for all cholesterol ester markers (CE), while none of the This signal was only observed in the non-affected tissue sur-
CE signals was found to be depleted relative to control rounding the granuloma. The corresponding ion was found 
samples. Cardiolipin markers (CL), on the other hand, as a marker that is enriched in bisex samples compared to 
were found depleted, while no detected CL was enriched control samples. Inside the granuloma, lysolipid metabolites 
relative to control samples. No characteristic distribu- of PE were found (red). Images B and D, obtained from 
tion patterns were found for these species (CE and CL) non-infected samples, do not show any localized enrichment 
in MALDI MSI measurements, which is either due to a of these markers that can be attributed to tissue structures. 
missing lateral specificity or an insufficient signal-to- This suggests that the eggs might accumulate certain lipid 
noise ratio in MALDI for these compounds at small spot species, for example, the tentatively identified PS(42:4) 
sizes (high lateral resolution). (Fig. 5). On the other hand, they appear to consume and 
Fig. 5  Comparison between liver 
sections of a bisex-infected hamster 
(A, C) and a healthy control sam-
ple (B, D). For A, m/z 500.275684, 
LPE(20:4), [M − H ]−  (red) 
was found to be a marker for 
granuloma; m/z  776.526946, 
MMPE(16:0_22:6), [M −  H]− 
(blue)  a marker for surround-
ing tissue; and m/z  866.592639, 
PS(42:4), [M −  H]−  (green) a 
marker for schistosome eggs. For 
the non-infected sample, B shows 
the same ions in the same colors, 
but no characteristic distribution 
was found. This underlines that 
statistical markers found by LC–
MS/MS also show recognizable 
and allocatable distributions in the 
imaging measurements. In image 
C, m/z  746.511353, plasmenyl-
PE(P-16:0/22:6), [M −  H]−  (red) 
as a marker for granuloma and 
m/z  752.555581, plasmanyl-
PE(O-18:0/20:4), [M −  H]−  (green) 
as a marker for granuloma borders 
were selected for creating the RGB 
image. Again, m/z 776.526946 was 
taken as a marker for non-affected 
tissue (blue). The same coding as 
in C was chosen for image D of a 
non-infected (control) sample, and 
again, we found no characteristic 
distribution. Scale bars are 1  mm. 
Single-ion images are shown in 
Figure  S7 and Figure  S8. E and 
F are the corresponding optical 
images of the samples
1 3
Changes in the lipid profile of hamster liver after Schistosoma mansoni infection,… 3661
thus downgrade the contents of certain lipids in the close at m/z 800.556617 as [M + K ]+, was found only in the 
vicinity of granulomas in bisex samples, as, for example, granulomas and just rarely in the surrounding tissue or in 
for MMPE (see single-ion images and optical images in not-infected samples (Figure S12). The according mass 
Figure S7, S8, and S14). Taking a look at the single-ion spectrum and extracted ion chromatogram are shown in 
images, “holes” in the distribution of granuloma-specific Figure S4. PC(20:0_20:3), detected at m/z 862.630305 
ions were observed. The microscopic images show that this as [M + N a]+, showed some enrichment in and directly 
is not due to missing tissue but schistosome eggs are found around the eggs, but not in the rest of the sample (Fig-
at these positions. This is a further hint that the eggs take up ure S13). These observations provide first hints about pos-
these lipids and metabolize them. However, as no labelled sible metabolic changes in the tissue due to infection.
tracer studies were carried out, this is only a hint and not a Another example is shown in Fig. 6. Here, the lateral 
clear proof. A deeper look into lipid uptake of schistosome distribution of TG(50:2), detected as [M + K ]+ ion at 
eggs is described in a recent study of von Bülow et al. [in m/z 869.69869, is shown. This lipid was detected at m/z 
preparation]. Of special interest are the two lipids shown 848.771097 as [M + N H ]+4  in LC–MS/MS experiments 
in the green color channel in Fig. 5A and C, which were and also found in eggs before by Giera et al. [21]. Our 
found in distinct areas of the granuloma only. The compound statistical LC–MS/MS analysis revealed that this TG was 
at m/z 752.555581 was identified by LC–MS/MS as a PE depleted in tissue of bisex-infected hamsters. MSI data 
lipid (plasmanyl-PE(O-18:0/20:4), [M −  H]−); the other revealed distribution changes due to infection. While the 
compound at m/z 866.592639 was tentatively assigned as lipid was found to be evenly distributed in the three control 
PS(42:4), [M − H ]−. Other examples can be found in Fig- samples, it was only found in the granuloma of the bisex-
ure S9. While some marker lipids were found within or in infection group. All images shown in Fig. 6 were adjusted 
direct contact to the eggs, others were located mostly in the to the same signal intensity. Therefore, pixel brightness 
outer regions of the granuloma (shown in the green color in all sub-images corresponds to the same signal intensity 
channel). Eggs have to take up nutrients from their host tis- scale. Bisex samples on the left-hand side show negligible 
sue to survive [43] and our MS image data seem to substanti- signal intensities of TG(50:2) except from the granuloma-
ate this assumption. The enrichment or depletion of specific tous areas, where signal intensities peaked to NL = 8*103. 
lipids might be related to the presence of cells forming the In the control samples, the lipid was found ubiquitously 
granuloma and therefore displacing the hepatocytes. We and signal intensities were in the range of NL = 1*103 to 
found no clear correlation between localization and an entire NL = 2.2*103. In control sample 1, signal intensities were 
lipid class but only with individual lipid species. lower compared to the other control samples, but the sig-
Additionally, the distribution pattern based on differ- nal was still evenly distributed throughout the whole tissue 
ent adducts for the same lipid was investigated. This can section.
be exemplarily seen in Figure S6. Here, the distribution This tendency was also observed for eight other triglyc-
of PC(O-34:1) is shown as proton, sodium, and potas- erides. Due to low signal intensities, however, the effect was 
sium adducts. In general, this lipid was mainly found in the not as obvious in some cases. For the triglycerides with the 
granuloma around but not in the eggs. However, on closer lowest signal intensities, accumulation of the respective lipid 
examination, signal intensities and localization outside the was observed in the granuloma of the bisex-infection group, 
granuloma region vary slightly. This can be due to different whereas detection in control samples was limited to only a 
distributions of the alkali metals in the tissue. In general, few pixels per sample. Interestingly, we have not observed 
signal intensities of some lipid classes (e.g., TGs) were not the opposite trend of a depletion in granuloma for any tri-
that high. After dividing low abundance of one compound glyceride in our MSI data. These data provide new insights 
to three different adduct signals, intensities might be too into the localization and accumulation of triglycerides in 
low for detection. Therefore, not all adducts were found for livers of Schistosoma-infected hamsters at the level of indi-
each lipid. vidual lipid species.
We have chosen the lipid class of PCs to take a deeper We also compared infection markers found in our 
look at the distributions of several lipid species. Inter- study with those found in schistosome eggs by Giera et al. 
estingly, different distribution patterns were found. For [21]. Tentative markers that we specifically found in eggs 
example, PC(16:0_20:5), observed at m/z  802.535727 (DMPE(18:0_22:5) and PS(42:4)) were not detected by 
as [M +  Na]+, was found to be evenly distributed in the Giera et al. or Kadesch et al., neither in eggs nor in schisto-
whole sample except in the granulomatous areas in liver some worms. Infection markers of the lipid classes CE, LPC, 
samples of bisex-infected hamsters, where it was depleted PC, and TG were in good accordance with previous data 
(Figure  S10). In contrast, PC(19:0_20:2), detected at [20, 21], suggesting that the eggs might take up the respec-
m/z 850.629627 as [M + N a]+, was found to be enriched tive lipids, perhaps as nutrients. Other markers, such as PEs, 
in granulomas (Figure  S11). PC(16:0_18:0), detected CLs, dimethylphosphatidylethanolamines (DMPEs), and 
1 3
3 662 Wiedemann K. R. et al.
Fig. 6  Upper part: Distribution comparison of m/z 869.698690 accumulation in the eggs and the surrounding granuloma area of the 
TG(16:0_16:1_18:1) [M +  K]+. Liver samples of bisex-infected ham- bisex sample group is clearly visible. Overall, this triglyceride was 
sters are shown on the left side; control samples are grouped on the found enriched in control samples by LC–MS/MS. Lower part: Cor-
right side. While the lipid is evenly distributed in the control samples, responding optical images of the samples. Scale bars are 1 mm
1 3
Changes in the lipid profile of hamster liver after Schistosoma mansoni infection,… 3663
diglycerides (DGs), seemed to be characteristic for the host mechanisms and the role of these lipids in the host-parasite 
tissue since they were not detected in eggs by Giera et al. interaction of S. mansoni.
Additional investigations are needed to get a better 
understanding of the underlying mechanisms of lipid Supplementary Information The online version contains supplemen-
localization, presumptive uptake mechanisms, and accu- tary material available at https://d oi.o rg/1 0.1 007/s 00216-0 22-0 4006-6.
mulation. These might be related to lipid consumption by 
Acknowledgements We thank TransMIT GmbH, Giessen, Germany, 
the eggs or to reactions of the host’s immune system. Other for technical support. We kindly thank Vannuruswamy Garikapati and 
studies have shown that granuloma protects the host from David Lüke for their help with the LC-MS/MS experiments as well as 
enzymes secreted by the eggs [7, 44], and the identified Christina Scheld and Georgette Stovall for excellent technical assis-
lipids in the outer regions of the granuloma might be part tance in maintaining the parasite cycle.
of this defense mechanism. Stanley et al. reported about 
Funding Open Access funding enabled and organized by Projekt 
lipid-enriched cells in the outer part of the granuloma and DEAL. This work received financial support from the Hessian Minis-
suggested that immune cells, such as macrophages, might try of Science, Higher Education and Art (HMWK), LOEWE Center 
play an important role in the changes of the lipid profile DRUID, and from Deutsche Forschungsgemeinschaft DFG (Sp314/13–
of the host’s liver [15]. According to our findings, only 1, INST 162/500–1 FUGG, and RO3714/4–1).
plasmanyl- and plasmenyl-PEs (plasmalogens) seemed 
to accumulate at the borders of granuloma (see Fig. 5). Declarations 
Plasmalogens were mainly found in the outer tegumen-
Ethics approval, source of biological material, and statement on ani-
tal membranes of adult worms [45]. This membrane is mal welfare To keep the S. mansoni life cycle, Biomphalaria glabrata 
the contact region between host and parasite and plays an snails served as intermediate hosts and Syrian hamsters (Mesocrice-
important role in defending the parasite against the host’s tus auratus) as final hosts. The hamster model has been established 
immune system [46, 47]. because it is more permissive for schistosome infection compared to the mouse model, which requires more animals (reduction principle 
The results of our study provide first insights into the of the 3Rs). Both snails and hamsters were bred in-house (Biomedical 
alteration of lipid profiles in hamster livers after infection Research Center Seltersberg, Giessen, Germany). All animal experi-
with S. mansoni and egg deposition. Especially the detailed ments were conducted in accordance with the European Convention for 
information on lipid species level that was achieved by the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS No 123; revised Appendix A), and they were 
LC–MS/MS analysis in connection with lateral information additionally approved by the Regional Council (Regierungspraesidium) 
gained through MALDI MSI will open novel perspectives to Giessen (V54-19 c 20/15 c GI 18/10).
unravel the roles of individual lipid species and lipid classes 
in the context of the egg-induced pathological changes in the Conflict of interest B.S. and C.G.G. are consultants of TransMIT 
host liver tissue. GmbH, Giessen, Germany. The other authors declare to have no con-
flicts of interest.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
Conclusion tion, distribution and reproduction in any medium or format, as long 
as you give appropriate credit to the original author(s) and the source, 
With the help of mass spectrometric techniques, we char- provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are 
acterized morphological changes, such as granuloma for- included in the article's Creative Commons licence, unless indicated 
mation in the liver tissue of S. mansoni-infected hamsters otherwise in a credit line to the material. If material is not included in 
at a molecular level. Alterations in the lipid profile were the article's Creative Commons licence and your intended use is not 
examined by LC–MS/MS of homogenized samples, and for permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a 
the first time, we identified markers specific for infection. copy of this licence, visit http://c reati vecom mons.o rg/l icens es/b y/4.0 /.
These markers were additionally analyzed by MALDI MSI, 
exhibiting their distribution in tissue sections. The estab-
lished protocol allowed not only to visualize and analyze 
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