Alsina-Sanchis et al., Oil depletes resident peritoneal macrophages
1
Intraperitoneal oil application causes local inflammation with depletion of 1
resident peritoneal macrophages 2
Elisenda Alsina-Sanchis
1
, Ronja Mülfarth
1
, Iris Moll
1
, Carolin Mogler
2
, Juan Rodriguez-Vita
1,*
, 3
Andreas Fischer
1,3,4,*
4
1
Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), 5
69120 Heidelberg, Germany. 6
2
Institute of Pathology, Technical University of Munich, 81675 Munich, Germany. 7
3
Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, 69120 8
Heidelberg, Germany. 9
4
European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, 68167 10
Mannheim, Germany. 11
*, These authors contributed equally to this work 12
13
Running title: Oil depletes resident peritoneal macrophages 14
Key words: oil; resolution of inflammation; peritoneum; thioglycolate; xanthogranuloma; 15
* Correspondence: Division of Vascular Signaling and Cancer, German Cancer Resersach 16
Institute, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Phone: +40 6221 4150. 17
FAX: 06221 42 4159. Email: j.rodriguezvita@dkfz.de (J. Rodriguez-Vita) and
18
a.fischer@dkfz.de (A. Fischer).
19
Funding sources: This work was funded by the Deutsche Forschungsgemeinschaft (DFG) 20
project number 394046768 - SFB1366 projects C4 and Z2 (to A.F., C.M.), DFG project number 21
419966437 (to J.R.V.), the Cooperation Program in Cancer Research of the Deutsches 22
Krebsforschungszentrum (DKFZ) and Israel’s Ministry of Science and Technology (MOST) Ca 23
178 (to A.F. and R.M.) and the Helmholtz Association (to A.F.). 24
Conflict of interest: The authors declare that they have no conflict of interest. 25
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Alsina-Sanchis et al., Oil depletes resident peritoneal macrophages
2
Abstract 26
Oil is frequently used as a solvent to inject lipophilic substances into the peritoneum of 27
laboratory animals. Although mineral oil causes chronic peritoneal inflammation, little is known 28
whether other oils are better suited. Here we show that olive, peanut, corn or mineral oil causes 29
xanthogranulomatous inflammation with depletion of resident peritoneal macrophages. 30
However, there were striking differences in the severity of the inflammatory response. Peanut 31
and mineral oil caused severe chronic inflammation with persistent neutrophil and monocyte 32
recruitment, expansion of the vasculature and fibrosis. Corn and olive oil provoked no or only 33
mild signs of chronic inflammation. Mechanistically, the vegetal oils were taken up by 34
macrophages leading to foam cell formation and induction of cell death. Olive oil triggered 35
caspase-3 cleavage and apoptosis, which facilitates the resolution of inflammation. Peanut oil 36
and, to a lesser degree, corn oil triggered caspase-1 activation and macrophage pyroptosis, 37
which impairs the resolution of inflammation. As such, intraperitoneal oil administration can 38
interfere with the outcome of subsequent experiments. As a proof-of-principle, intraperitoneal 39
peanut oil injection was compared to its oral delivery in a thioglycolate-induced peritonitis 40
model. The chronic peritoneal inflammation due to peanut oil injection impeded the proper 41
recruitment of macrophages and the resolution of inflammation in this peritonitis model. In 42
summary, the data indicate that it is advisable to deliver lipophilic substances like tamoxifen 43
by oral gavage instead of intraperitoneal injection. 44
45
Introduction 46
Oil is frequently used as solvent in animal research. For instance, inducible gene 47
recombination using the Cre-ERT2 -loxP system requires administration of tamoxifen which is 48
usually dissolved in olive, peanut, corn or mineral oil. The oil solution is administered orally or 49
by intraperitoneal injection (i.p.) (1, 2). Also, in a liver fibrosis model carbon tetrachloride (CCl
4
) 50
is delivered by i.p. injection in oil, inhalation or oral gavage (3). Interestingly, i.p. injection 51
generates stronger liver fibrosis when compared with the other two administration methods (4), 52
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Alsina-Sanchis et al., Oil depletes resident peritoneal macrophages
3
raising the question whether CCl
4
or its solvent act locally within the peritoneum. Indeed, i.p. 53
injection of mineral oil causes chronic inflammation (5-9). Also, subcutaneous injection of olive 54
oil can cause lipogranuloma, a granulomatous inflammatory soft tissue reaction (10). 55
Therefore, it can be assumed that any experimental immune cell analysis within the 56
peritoneal cavity would be strongly affected by oil. It is surprising how little is known about the 57
peritoneal immune cell reaction towards oil and comparative studies of different oils are 58
missing to our knowledge. 59
Peritoneal inflammation can be divided into the initiation and resolution phase. 60
Pathogens trigger infiltration of neutrophils, which phagocytose pathogens, clear apoptotic 61
cells and recruit monocytes from the blood stream into the peritoneal fluid. Recruited 62
monocytes eliminate dying neutrophils and differentiate into monocyte-derived macrophages 63
(11). This is important, as the number of resident peritoneal macrophages, which are derived 64
from embryonic progenitors and have self-renewal capacity (12), get strongly decreased as a 65
result of the so-called “macrophage disappearance reaction” (13). As such, resident peritoneal 66
CD11b
+
macrophages, expressing high F4/80 levels (F4/80
hi
) get replaced by monocyte-67
derived CD11b
+
macrophages, expressing low F4/80 levels (F4/80
low
) on the membrane (12, 68
14, 15). Subsequently, monocyte-derived macrophages increase surface expression of F4/80 69
from a low to an intermediate level (F4/80
int
) to initiate the resolution phase (16). 70
The switch from inflammatory to resolving macrophages is triggered by phagocytosis 71
of apoptotic cells. Deficiency in this phagocytic process leads to chronic inflammation (17). For 72
instance in atherosclerotic plaques, macrophages take up excessive amounts of lipids and 73
become foam cells, which cannot initiate the resolution phase, perpetuating further neutrophil 74
and monocytes infiltration (18). 75
The aim of this study was to analyze how the most commonly used oils in animal 76
research affect the myeloid cells within the peritoneum and whether this would diminish their 77
capability to resolve the peritoneal inflammation. 78
79
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Alsina-Sanchis et al., Oil depletes resident peritoneal macrophages
4
Materials and methods 80
Animal models 81
The study was approved by institutional and regional animal research committees. All animal 82
procedures were in accordance with institutional guidelines and performed according to the 83
guidelines of the local institution and the local government. Female C57BL/6 mice were group‐84
housed under specific pathogen‐free barrier conditions. 85
Administration of peanut (P2144, Sigma-Aldrich, St. Louis, USA), corn (C8267, Sigma- Aldrich, 86
St. Louis, USA), olive (88631, Carl Roth, Germany), mineral oil (HP50.2, Carl Roth, Germany) 87
or 0,9% sterile NaCl (Braun, Germany) in 8 to 12-week-old randomized mice was performed 88
by daily i.p. injection of 100 µl for 5 consecutive days or by oral gavage of peanut oil once with 89
100 µl. After three weeks mice were euthanized. For peritoneal lavage, 5 ml of cold PBS 90
(Gibco/Thermo Fisher Scientific, NY, USA) was injected i.p. after a careful massage to mobilize 91
cells, peritoneal fluid was collected. Cells were isolated by centrifugation (5 min, 200 g) and 92
suspended in 1 ml of PBS. 93
8 to 12-week-old randomized mice were euthanized and administrated with peanut, olive, corn 94
and mineral oil. After 5 minutes the peritoneal lavage was collected. 95
Three weeks after oil treatment, mice were i.p. injected with thioglycolate (2 mg in 1 ml H
2
O; 96
B2551, Sigma Aldrich, St. Louis, USA). After 24 or 72 hours mice were sacrificed and 97
peritoneal lavage collected. All groups were randomized. 98
99
Immunofluorescence and tissue histology 100
Histological analysis was performed on formalin‐fixed paraffin‐embedded sections (3 μm). 101
Sections were deparaffinized and rehydrated. For hematoxylin-eosin (H&E) and Sirius Red 102
(Dianova, Germany) staining, sections were processed according to standard protocols. For 103
myeloid cell staining, antigen retrieval at pH 6 with citrate buffer and the primary antibody rabbit 104
anti-mouse CD11b (1:200) (ab133357, Abcam, Cambridge, MA, USA) and antigen retrieval 105
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Alsina-Sanchis et al., Oil depletes resident peritoneal macrophages
5
with 1:20 proteinase K/TE buffer and rat anti-mouse F4/80 (1:100) (T-2006, Dianova, 106
Germany) incubated at 4°C overnight. After washing, sections were incubated with secondary 107
antibodies coupled with HRP (1:200) (DAKO, Agilent Technologies, Santa Clara, CA, USA) for 108
one hour at room temperature. For immunofluorescence staining, antigen retrieval at pH 9 was 109
performed using citrate buffer and sections were incubated with the primary antibody rabbit 110
anti‐mouse CD31 (1:50) (ab28364, Abcam, Cambridge, MA, USA) at 4°C overnight. After 111
washing, sections were incubated with secondary antibody (1:200) goat anti‐rabbit Alexa 112
Fluor-647 (A21245, Life Technologies/Thermo Fisher Scientific, NY, USA) for 1 hour at room 113
temperature. H&E images were obtained with slide scanner (Zeiss Axio Sacn.Z1, Carl Zeiss, 114
Germany). CD11b images were obtained with widefield microscope (Zeiss Axioplan, Carl 115
Zeiss, Germany). All images were processed with ZENblue software (Carl Zeiss, Germany). 116
Immunofluorescence was imaged at the confocal (LSM 700, Carl Zeiss, Germany) microscope 117
with ZENblack software (Carl Zeiss, Germany). Sections of seven Z-stacks per omentum and 118
mesentery and three random fluorescence images per slide were taken. Numbers of CD31 119
positive vessels per view field and lipid droplet size from H&E images were counted with 120
ImageJ software (NIH, Bethesda, MD, USA). 121
122
Oil Red O staining 123
Peritoneal lavage was plated into one well of a 6-well plate on top of coverslips and incubated 124
for 30 min with Dulbecco’s modified Eagle’s medium (DMEM) (Gibco/ Thermo Fisher Scientific, 125
NY, USA). Afterwards non-adherent cells were removed by careful washing three times with 126
PBS. J774A.1 cells cultured in DMEM with 10% fetal calf serum (Biochrom, UK) were seeded 127
into 12-well plates on coverslips and treated with 100 µl oil in 1 ml medium for four hours. Cells 128
on coverslips were stained with Oil Red O (O0625, Sigma-Aldrich) following the protocol 129
published elsewhere (19) and counterstained with hematoxylin. Images were obtained with 130
widefield microscope (Zeiss Axioplan Carl Zeiss, Germany). 131
132
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The copyright holder for this preprintthis version posted July 29, 2020. ; https://doi.org/10.1101/2020.07.15.203885doi: bioRxiv preprint
