




































_____________________________________________________________________________________________________ 
 
*Corresponding author: E-mail: hafr@sund.ku.dk; 
 
 
 

Asian Journal of Immunology 
 
4(1): 160-174, 2021; Article no.AJI.83640 
 

 
 

 

 

High Fat Diet Triggers a Prompt and Transient 
Increase in Adipose Tissue Granulocyte Colony 

Stimulating Factor and Circulating Myeloid  
Cells in Mice 

 
Helene M. S. Eld a, Louise Madsen a, Christina H. Lund a,  

Stine Broeng Metzdorff a and Hanne Frøkiær a* 
 

a 
Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University 

of Copenhagen, Ridebanevej 9 st, 1870 Frederiksberg C, Denmark. 
 

Authors’ contributions  
 

This work was carried out in collaboration among all authors. All authors read and approved the final 
manuscript. 

 
Article Information 

 
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Received 25 October 2021  
Accepted 28 December 2021 
Published 30 December 2021 

 
 

ABSTRACT 
 

Scope: The short-term effects of feeding high fat diet (HFD) to mice was investigated with focus on 
the effect on myelopoesis, circulating neutrophils and the induction of Granulocyte colony 
stimulating factor (G-CSF).  
Methods: Male mice were fed HFD (45%) during a period of 5 weeks with samples taken after 3 
days and 1, 3, 4 and 5 weeks. Blood was analyzed for neutrophils and monocytes, for G-CSF and 
granulocyte-macrophage (GM)-CSF, and for cytokine expression. Visceral adipose tissue (VAT) 
expression of various genes and production of G-GSF and GM-CSF in cultured VAT was 
determined. 
Results: Three days after commencement of HFD, the number of circulatory neutrophils and 
monocytes increased but returned to baseline-level at day 8. This transient increase coincided with 
an increased blood concentration of G-CSF and a transient increase in bone marrow and spleen 
neutrophils. In supernatant from cultivated visceral adipose tissue isolated from HFD fed mice on 
day 3 and 8, G-CSF was increased. The expression of Toll-like receptor 4 in adipose tissue was 
down-regulated from week 4. In vitro, lipopolysaccharide (LPS) was a poor stimulator of G-CSF, 

Original Research Article 



 
 
 
 

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161 

 

while G-CSF or LPS together with G-CSF or GM-CSF induced increased G-CSF production. G-

CSF suppressed production of LPS-induced TNF and increased IL-10 production in dendritic cells 
suggesting that G-CSF down-regulates LPS-induced inflammation.  
Conclusion: HFD induces a transient increase in adipose tissue G-GSF and circulating myeloid 
cells in mice. We suggest G-CSF induces increased myelopoiesis and simultaneously down-
regulates LPS-induced inflammation. 
 

 
Keywords:  Adipose tissue; granulocyte-coloni stimulating factor (G-CSF) production; high fat diet; 

monocytes; neutrophils. 
 

1. INTRODUCTION  
 

Daily or regular intake of high fat meals is 
common in large parts of the world and may lead 
to overweight. Alongside the weight gain, a state 
of systemic low-grade inflammation (LGSI) often 
emerges, which in the long run may lead to the 
development of metabolic syndrome and further 
on, to chronic diseases such as type 2 diabetes 
(T2D) and cardiovascular diseases [1]. However, 
the precise relationship between high fat diet 
(HFD) consumption, inflammation and obesity, is 
still not fully understood. Also consumption of a 
single high fat meal has been reported to induce 
an inflammatory response and has been 
suggested to jumpstart the low-grade 
inflammation [2]. A systematic review on the 
postprandial inflammatory response to a high fat 
meal in healthy adults did only reveal an effect 
on blood IL-6 but not on other pro-inflammatory 
markers and the 47 studies included in the 
review revealed large variations in the post-
prandial inflammatory response [2]. The reason 
for this variation remains obscure. Very little has 
been described regarding the effects of the early 
course of a HFD before the onset of diet-induced 
weight gain and obesity as regard the 
inflammatory events taking place in bone 
marrow, adipose tissue and blood, with potency 
to jumpstart the development of LGSI and 
comorbidities.  

 
Microbiota has been suggested to play a role in 
HFD-induced inflammation via its production of 
endotoxin [3, 4]. Increased dietary lipid increases 
the lymphatic flow and leads with it an increased 
amount of endotoxin from the gut [5, 6].

 
The full 

consequences of increased endotoxin influx are 
however not fully understood. The effect of an 
LPS injection on granulopoiesis and increased 
G-CSF levels is well established [7, 8], but the 
consequences of a continuous LPS influx from 
the gut as expected from a permanent or regular 
HFD have only been sparsely reported. Cani et 
al. showed that a HFD fed to mice for four weeks 
lead to an increase in the circulatory endotoxin 
level and provided evidence that LPS was 

responsible for the onset of metabolic disease 
leading to T2D in mice [9].  Apart from LPS, also 
free fatty acids (FFA), in particular saturated FFA 
stimulate inflammation through the binding to 
TLR4 [10]. Hence, as both FFA and LPS influxes 
are presumed to be increased in HFD fed mice 
both may contribute in the stimulation of an 
inflammatory response [11, 12]

 
indicating that 

solely measuring the LPS concentration in e.g. 
blood, may not directly correlate with an 
inflammatory response. 
 
Mice fed a diet rich in fat (45-60%) serve as a 
frequently used model for studying the 
physiological effects of an excess energy intake 
and obesity. Obesity and firmly established LGSI 
typically emerge after HFD feeding for eight 
weeks or more whereas comorbidities such as 
T2D establish even later [13]. The inflammatory 
response is complex and studies of the 
relationship between obesity and physiological 
changes have revealed that a connection 
between visceral adipose tissue (VAT), 
hematopoiesis (bone marrow) and blood exists 
[14, 15]. Whether shorter periods of feeding HFD 
and inflammation exhibit similar relationships is 
unclear.  
 

Talukdar et al. showed that as early as three 
days after the onset of feeding a HFD, neutrophil 
infiltration in VAT, which remained high during 90 
days of feeding was seen [16]. Others have 
reported a transient neutrophil infiltration in the 
VAT ceasing one week after the onset of feeding 
a HFD [17]. Hence, neutrophil influx in VAT does 
take place, however the length of this event and 
how it is effectuated and perhaps abrogated 
remains obscure. Due to the short lifespan of 
neutrophils, a constant infiltration into adipose 
tissue requires a constant supply of neutrophils 
from the bone marrow. An increase in circulatory 
neutrophils may thus require a steady 
mobilization signal and an increased production 
in the bone marrow. 
 

Here, we investigated the effect of short-term 
feeding a HFD to young mice on bone marrow 



 
 
 
 

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162 

 

and blood cell composition and further assessed 
the circulatory and VAT level of G-CSF and GM-
CSF as well as the expression of genes involved 
in LPS-induced inflammation in VAT. We found 
that a transient neutrophil infiltration in VAT 
coincided with a transiently increased VAT 
production of G-CSF. We suggest that G-CSF 
holds dual activity stimulating neutrophil 
recruitment while simultaneously suppressesing 
inflammatory responses of other myeloid cells.  
This may explain the transient nature of 
myelopoiesis and advocates against LPS as a 
key initiator of HFD-induced LGSI [9]. 
 

2. MATERIALS AND METHODS 
 

2.1 Description of the Study Area 
 
2.1.1 Animals and feeding regime used in the 

study 
 
Male C57BL/6 mice (Taconic, Lille Skensved, 
Denmark) 6 weeks of age, were housed with 4 
mice per cage and fed ad libitum either a high-fat 
diet (HFD) (Altromin C-1000 rodent diet modified 
as HFD with 45% energy from fat, Brogaarden, 
Denmark) or a standard rodent diet with 5% kcal 
from fat (Altromin C-1000, Brogaarden). At study 
day 1, all mice were weighed and allocated to 
HFD (40 mice) or the standard diet (20 mice). 
The mice were euthanized by cervical dislocation 
and tissue samples collected under sterile 
conditions. The experiment was carried out in 
accordance with the Council of Europe 
Convention European Treaty Series 123 on the 
Protection of Vertebrate Animals used for 
Experimental and Other Scientific Purposes, and 
the Danish Animal Experimentation Act (LBK 
1306 from November 23, 2007) approved by the 
Animal Experimentation Inspectorate, Ministry of 
Justice, Denmark (License number: 2012-15-
2934-00256  C1-6). 
 

2.2 Method of Sample and Data Collection  
 
At day 3, 8, 23, 30 and 37, eight mice fed HFD 
and four fed standard diet were weighed and 
anesthetized. After blood sampling, the mice 
were euthanatized. Whole blood was collected 
from the retro-orbital vein with heparin-coated 
capillaries into EDTA-coated eppendorf tubes. 
Thirty μL blood was transferred to cryotubes 
containing 180 μL lysis buffer (50% Lysis/Binding 
Solution Concentrate, 50% isopropanol, Thermo 
Fisher, Waltham, MA, USA) and stored at -80°C 
until RNA extraction. The remaining blood was 
used for complete blood count (CBC) and 

leukocyte differentiation and isolated plasma was 
transferred and frozen at -80°C until ELISA was 
performed. Immediately after euthanasia, spleen, 
epididymal (visceral)  adipose tissue (VAT) and 
femur were collected. The spleen was cut in two 
and one part was placed in RNAlater (Ambion by 
Life Technologies, Thermo Fisher) for qPCR and 
the other part was placed in cold sterile PBS for 
cell isolation and flow cytometry. Bone marrow 
(BM) cells were isolated from the femur by 
flushing them out from cleaned bones with cold 
PBS followed by flow cytometry analysis [18]. 
The VAT was isolated and placed in a pre-
weighed tray with ice-cold PBS and weighed. 
Hereafter, a small part of the VAT was placed in 
RNAlater for qPCR and the rest used for 
culturing.  
 

2.3 Cell Isolation and Flow Cytometry 
Analyses 

 
Analyses on full blood was performed without 
any purification. Bone Marrow  and spleen cells 
were isolated as described previously

 
[18]. Blood 

neutrophilic granulocytes and monocytes 
proportions were assessed by blood leukocyte 
counting by an automatic cell counter (Advia 
2120i Hematology System, Siemens, Germany). 
To analyze single cell suspensions of spleen and 
BM cells by flow cytometry, cells were counted, 
washed and suspended in FACS wash buffer 
(PBS, 1% fetal bovine serum). For the 
identification of neutrophils and mature 
monocytes, a cocktail of anti-mouse Ly6G (1A8), 
Ly6C (AL-21), CD11b (M1/70) and CD115 (T38-
320) antibodies were used (BD Pharmingen, BD 
Biosciences, San Jose, CA, USA) [18]. For the 
identification of mature myeloid cells, Fc-
receptors were first blocked with Fc-block (BD 
Pharmingen, BD Biosciences) for 10 min at 4°C, 
washed in FACS wash buffer and incubated with 
the antibody cocktail at 4°C for 30 min. After 
washing with FACS wash buffer, cells were 
analyzed using a BD FACS CANTO II (BD 
Biosciences). Data were analyzed with FlowJo

TM
 

software (BD Biosciences) and the number of 
neutrophils and monocytes was calculated from 
total number of cells for each sample.  
 

2.4 RNA Isolation and qPCR  
 
Adipose tissue was homogenized in lysis buffer 
(MagMAX-96 RNA Isolation Kit; Ambion, Thermo 
Fisher) by using glass beads and the The 
FastPrep®-24 Instrument (MP Biomedicals, 
Thermo Fisher). Total RNA from homogenized 
adipose tissue, BM cells and blood cells was 



 
 
 
 

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163 

 

extracted by MagMAX Express (Applied 
Biosystems, Foster City, CA, USA) using the 
MagMAX-96 RNA Isolation Kit (Ambion, Thermo 
Fisher) for tissues and the MagMAX-96 Blood 
RNA Isolation Kit (Ambion, Thermo Fisher) for 
blood cells, as previously described [19] and 
according to manufacturer’s instructions. cDNA 
was produced from ~200 ng total RNA using 
High-Capacity cDNA Reverse Transcriptase Kit 
(Applied Biosystems) according to the 
manufacturer’s instructions. Gene expression of 
selected genes listed in Table 1 was analyzed on 
the StepOnePlus instrument (Applied 
Biosystems) using universal fast thermal cycling 
parameters (Applied Biosystems) and TaqMan 
Fast universal PCR Mastermix (Applied 
Biosystems). Relative quantification of the gene 
expression was calculated by the comparative 
cycle threshold (CT) method. The expression of 
target genes was normalized to the gene 
expression of Actb (beta-actin) as the reference 
gene: [∆CT =CT (target) –CT (reference)]. Relative 
quantification of gene expression was calculated 
as 2

-∆∆CT
, where ∆∆CT = [∆CT (sample)-∆CT 

(calibrator)]. Mean ∆CT of samples from control mice 
was used as the calibrator. 
 

2.5 Culturing of Adipose Tissue 
 
The epidydimal fat pads (VAT) were washed in 
PBS and two 50 mg pieces were placed in two 
different wells of a 6-well-plate and cut into 5 
smaller pieces and cultured for 24 hours in 2 ml 
M199 media with glutamine, 25 mM HEPES and 
1% pen/strep [20]. After culturing the adipose 
tissue supernatant from each mouse was frozen 
at -80°C until cytokine quantification.  
 

2.6 Cytokine Quantification  
 
Plasma G-CSF and G-CSF, GM-CSF, CXCL-2, 

TNF, IL-10 and IL-1β in the supernatant of 
cultured adipose tissue and of stimulated cells 
were quantified by DuoSet ELISA kits from  R&D 
Systems (MN, USA) according to manufacturer’s 
instructions.  
 

2.7 In vitro Treatment of Murine Bone 
Marrow Cells  

 
Bone marrow cells were isolated as described 
elsewhere [21]. Briefly, bone marrow from 
C57BL/6 mice was flushed from the femur and 
tibia and washed twice in sterile PBS. Cells 
(2.9×10

6
) were seeded in 12-well-plates in 1 mL 

RPMI 1640 (Sigma-Aldrich, St. Louis, MO), 

containing 10% (v/v) heat-inactivated fetal calf 
serum, penicillin (100 U/ml), streptomycin (100 
µg/ml), glutamine (4 mM) and 50 µM 2-
mercaptoethanol. Cells were then treated with 
LPS (Sigma-Aldrich), mouse G-CSF (R&D 
Systems) and/or GM-CSF (from a GM-CSF-
transfected myeloma cell line [21] in a final 
concentration of 0.1 µg/ml LPS, 10 ng/ml G-CSF 
and GM-CSF. The cells were incubated for 1 or 4 
days at 37°C in a 5% CO2 humidified 
atmosphere. BM cells were harvested and 
examined for neutrophils and mature monocytes 
using flow cytometry and the concentration of 
cytokines in supernatant was determined by 
ELISA. Dendritic cells were prepared as 
described [21]. 
 

2.8 Statistical Analysis 
 

Statistical analysis was performed in GraphPad 
Prism version 5.03 (GraphPad Software). 
Significance was evaluated by 2-way ANOVAs, 
1-way ANOVAs or t-tests to determine 
differences between treatment groups and time 
points.  Relative quantification (RQ) is plotted in 
gene expression experiments and statistical 
analysis was performed on ∆CT values. In 
experiments where control groups showed 
constant expression level all data from control 
mice were pooled.  
 

3. RESULTS 
 

3.1 High Fat Diet-Increased Body and 
Epididymal Fat Pad Weight is 
Preceded by a Transient Increase in 
Blood Myeloid Cells in Mice  

 

Male mice aged 6 weeks at the start of the 
experiment, were fed either a 45% HFD or a 
control diet with 4% fat. The mice were weighed 
regularly during the 37 days feeding period. Both 
groups gained weight during the first three 
weeks, indicating that the mice were not fully 
outgrown at the beginning of the experiment [22]. 
At Day 30 and 37, the HFD mice had increased 
their body weight and the weight of the 
epididymal fatpads significantly compared to the 
control mice (Figs. 1a-b). Percentages of blood 
monocytes and neutrophils from the mice as 
determined by CBC revealed a constant 
percentage of monocytes and granulocytes in the 
control group (0.6% and 10.5%, respectively), 
(Figs. 1c-d). In the HFD group, at Day 3, the level 
of monocytes increased more than twofold, but 
decreased to the base-level at Day 8 (Fig. 1c). 
Also the level of granulocytes showed an 



 
 
 
 

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164 

 

indication of an increase at Day 3, this was 
however not significant (p=0.16). When 
analyzing the concentration of the growth factor 
G-CSF in the plasma, the control groups showed 

a fairly constant level around 75 pg/ml. In the 
HFD group at Day 3, the G-CSF concentration 
was increased but dropped thereafter to that of 
the control mice (Figs. 1e). 

 
Table 1. Taqman assays used for gene expression analysis 

 
Gene Gene name Assay ID 

Actb Actin, beta Mn00607939_s1 
Elane 
Arg1 
Ccl2 

Neutrophil Elastase  
Arginase 1 
Chemokine (C-C motif) ligand 2 

Mm01168929_g1 
Mm00475988  
Mm00441242_m1 

Cxcl2 Chemokine (C-X-C motif) ligand 2 Mm00436450_m1 
Hp Haptoglobin Mm00516884_m1 
Stfa2l1 Stefin A2 like 1 Mm04212095_mH 
Foxp3 Forkhead box P3 Mm00475162_m1 
Tlr4 Toll-like receptor 4 Mm00445273_m1 
S100A8 
Itgam/CD11b 
CD14 
CD8a 
F4/80 

S100 calcium binding protein A8 (calgranulin A) 
Integrin alpha M 
Cluster of Differentiation 14 
Cluster of Differentiation 8 a 
EGF-like module-containing mucin-like hormone receptor-like 1 

Mm00496696_g1 
Mm00434455  
Mm00438094  
Mm01182107-g1 
Mm00802529_ml 

 

 
 

Fig. 1. HFD induces a prompt transient rise in myeloid cells in the blood which is absent at the 
first signs of weight gain. A: Body weight and B: VAT weight in mice fed high fat diet (HFD) or 

chow for 37 days (HFD n=8, control n=4 for each time point). Two-way ANOVA performed. 
Percentages of C: neutrophils and D: monocytes in blood as determined by CBC. All Control 

groups pooled n=20. One-way ANOVAs performed. E: Concentration of G-CSF in blood 
determined by ELISA. One-way ANOVA performed. All data is shown as mean +/-SEM, 

***<0.001, **<0.01, *p<0.05 



 
 
 
 

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165 

 

3.2 Myelopoiesis is Transiently 
Increased Early in the High Fat Fed 
Feeding Period  

 

We found no differences between the HFD and 
the control group in the proportions of myeloid 
progenitor cells (Supplementary Fig. 1). The fully 
differentiated myeloid cells (gating strategy in 
Fig. 2a), however, exhibited increased level of 
neutrophils (Ly6G

+
Ly6C

+
CD11b

+
) at Day 8 

(1.9·10
7
 vs 1.4·10

7
 per BM, p=0.012) but 

returned to that of the control mice at Day 23 
(Fig. 2b). At day 37, a significant increase in 
neutrophils was seen in the HFD group 
compared to control (1.6·10

7
 vs. 1.3·10

7
, 

p=0.03). Also the monocyte population (Ly6G
-

Ly6C
+
 CD11b

+
CD115

+
) in the HFD group 

showed a trend towards increased numbers at 
Day 8 (p=0.18) and Day 23 (p=0.09) but not at 
later time points. Of note, from day 30 the 
proportion of monocytes increased markedly in 
both groups, presumably due to the outgrowth of 
the mice at this age. A number of genes 
including Elane, Arg1, Haptoglobin (Hp) and 
Itgam (CD11b), are expressed in neutrophils at 
specific differentiation stages (Fig. 2d) [23]. The 
expression of these genes in the BM cells was 
analyzed. In line with the increased neutrophil 
population detected in BM at day 8, a significant 
increase in the expression of Hp and CD11b 
indicative of neutrophils in the late differentiation 
state or mature neutrophils was detected at Day 
8 (Fig. 2e). No differences between the two 
groups were detected in the expression of Arg1 
or Elane. Thus, HFD causes a prompt but 
transient increase in mature myeloid cells in the 
BM.   
 

3.3 Spleen Myeloid Cells are transiently 
increased Early in the HFD Feeding 
Period  

 

When analyzing the myeloid population in the 
spleen during the course of HFD, we saw a non-
significant increase in neutrophils 
(Ly6G

+
Ly6C

+
CD11b

+
) at Day 8 (p=0.081) 

compared to control group (Fig. 3b).  Mature 
monocytes (Ly6G

-
Ly6C

+
 CD11b

+
CD115

+
) 

showed a trend towards increased numbers in 
the HFD group at Day 3 and Day 23 (p=0.11 and 
p=0.12, respectively) (Fig. 3c). These data 
support the finding from the blood leukocyte 
counting (Fig. 1c and d) showing that an early 
increase in blood neutrophils is induced upon 
HFD feeding. 
 
Expression analysis of blood was performed on 
Hp to identify immature neutrophils, on Il1b and 

Stfa2l1 to identify mature neutrophils, and Cd14 
to identify monocytes. The expression of Hp  
showed a tendency to be increased on Day 3 
(p=0.15) in HFD fed mice while on Day 8 
(p=0.061) and Day 23 (p=0.019) the expression 
dropped to become lower than the chow fed mice 
and then increased again on day 37 to the level 
of the control group (Fig. 3d). In the HFD group, 
the expression of Il1b and Stfa2l1 was 
significantly increased on Day 8 compared to the 
control group, while from Day 23 the expression 
of these genes was not different from the control 
group. The expression of Cd14 showed a 
significantly higher level at Day 23 compared to 
control group, while the expression did not 
significantly differ from the control group at any 
other day. Hence, during the first week of HFD 
feeding, the number of mature neutrophils in 
circulation transiently increases but drops 
subsequently to the level of the chow fed control 
group. These data indicate an early but transient 
recruitment of neutrophilic granulocytes from the 
bone marrow to the blood upon the onset of HFD 
feeding. 
 

3.4 Cultured Adipose Tissue from Mice 
at the Onset of High Fat Feeding 
Secretes Elevated G-CSF  

 

To investigate if adipose tissue could contribute 
to the enhanced G-CSF level measured in the 
blood at Day 3, VAT isolated from mice fed HFD 
or control diet were cultured for 24 hours and the 
concentration of released G-CSF and GM-CSF 
was measured in the supernatant (Fig. 4a-b). 
Adipose tissue from mice on control diet showed 
a constant production of G-CSF and GM-CSF 
and data from different time points was therefore 
pooled. The G-CSF concentration from cultured 
HFD VAT showed on Day 3 a trend to be 
increased (p=0.08) and at Day 8 a significantly 
increased G-CSF production (p=0.01) (Fig. 4a).  
Adipose tissue from 23, 30 and 37 days did not 
show increased G-CSF production. The HFD did 
not result in a changed GM-CSF production, 
which showed a stable level around 0.5 ng per 
gram cultured adipose tissue during the entire 
experiment (Fig. 4b). Also the concentration of 

IL-1 was analyzed but the concentration in all 
samples was at or below the detection limits 
(data not shown). 
 

3.5 Pro-Inflammatory as well as Anti-
Inflammatory Events Occur in 
Adipose Tissue of High-Fat Fed Mice   

 

To investigate which factors were induced in 
adipose tissue during the five weeks of HFD 



 
 
 
 

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166 

 

 
 

Fig. 2. High fat feeding reveals an early and transient up-regulation of neutrophils in bone 
marrow. A: Gating strategy for identification of neutrophils and monocytes in isolated bone 

marrow cells. B: The number of neutrophils (Ly6C
+
Ly6G

+
CD11b

+
) and C: monocytes 

(Ly6C
+
Ly6G

-
CD11b

+
CD115

+
) in bone marrow from HFD fed mice and control mice, HFD n=8, C 

n=4, individual unpaired t-tests performed. D: Schematic overview over the expression of 
selected genes in various differentiation stages of neutrophil granulocytes. E: Expression of 
genes at different time points in the HFD fed group (n=8) compared to control mice (n=4) by 

unpaired t-tests performed on dCt values. Mean+/-SEM, **p<0.01, *p<0.05 



 
 
 
 

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feeding, the expression of different genes was 
analyzed in the VAT of HFD fed and control mice 
(Fig. 5). For the control group, all the genes 
tested showed a stable expression during the 
entire experiment, accordingly expression data 
from the various days were pooled. In the HFD 
group, the expression of Ccl2 encoding the 
monocyte-recruiting chemokine CCL2 [24] was 
slightly increased at Day 8 (p=0.065) compared 
to control and was increased at Day 23, Day 30 
and 37 (Fig. 4). The expression of Cxcl2 
encoding the neutrophil-recruiting chemokine 
CXCL2 first showed a decreased expression on 
Day 3 (p=0.08) and then returned to the control 
level from Day 8 though with an increased 
expression at Day 30. Hence, the chemokines 
exhibited distinct expression profiles indicating a 
gradual increase of the monocyte-recruiting 
CCL2 and an initial reduction of the neutrophil-
recruiting CXCL2. The expression of Hp was 
transiently up-regulated at Day 3 and Day 8, 
while the expression of Stfa2l1 transiently 
showed a tendency to be increased on Day 8 
(not significant). The expression of F4/80 
signifying the presence of macrophages [25] did 
not at any time point deviate from the expression 
of the control group, while S1008a expressed 
constitutively by monocytes and neutrophils [26] 
showed an up-regulation at Day 30 and 37. 
While the expression of Cd8a did not at any time 
point differ from the control group, the expression 
of FoxP3 increased at the end of the study (Day 
37). Also the expression of TLR4 was analyzed 
and showed a significantly decreased expression 
at Day 30 and 37 compared to the control group. 
 

3.6 G-CSF Stimulates the Production of 
G-CSF in BM Cells But Inhibits an 
Inflammatory Response in Monocyte-
Derived Cells 

  

To investigate how G-CSF, alone or in concert 
with LPS affected the development of myeloid 
cells, LPS was added to freshly isolated BM 
cells, with or without concomitantly added G-CSF 
or GM-CSF, and incubated for 1 or 4 days, 
where after the cellular composition was 
assessed by flow cytometry (Fig. 6a-b). After 1 
day of stimulation with G-CSF or GM-CSF, the 
number of neutrophils doubled compared to cells 
cultured in media only, and after 4 days 
neutrophils were almost tripled compared to 
media-grown cells at day 1 (Fig. 6a).  The 
number of monocytes also increased in the 
presence of G-CSF or GM-CSF, after 1 day to 
1.5-2 fold and after 4 days to 4-6 fold the number 
in media. In the presence of LPS, the number of 

neutrophils at day 1 and 4 gradually dropped to 
around 60 and 20% of the media Day 1 control. 
Likewise, when LPS was added together with 
either G-CSF or GM-CSF, the levels of 
neutrophils reached about 75% of the level 
obtained without LPS at day 1 and, at day 4, the 
number of granulocytes dropped to below the 
Day 1 media control level. In contrast, LPS 
stimulated the development of monocytes 
leading to more than the doubled number of 
monocytes at Day 4 compared to media controls 
at day 1 and 4 (Fig. 6b). LPS added together with 
G-CSF or GM-CSF did not lead to further 
increase in the number of monocytes as 
compared to stimulation with G-CSF or GM-CSF 
alone.  
 
Neutrophils have a short lifespan, thus it is not 
possible from this experiment to establish 
whether LPS stimulates a faster development 
and thereby deplete the number of cells or halts 
the differentiation of these cells. To establish 
whether LPS stimulation affected the production 
of G-CSF or GM-CSF in the isolated bone 
marrow cells, which could represent an indirect 
way to stimulate myelopoiesis, we tested the 
concentration of G-CSF and GM-CSF in the 
supernatant of the stimulated bone marrow cells. 
LPS alone did not induce production of G-CSF, 
but addition of G-CSF led a strong production of 
G-CSF, and G-CSF together with LPS further 
increased the concentration (Fig. 6c). Compared 
to Day 1, the G-CSF level at Day 4 measured 
after addition of G-CSF alone or in combination 
with LPS decreased indicating that the G-CSF is 
spend during the development of neutrophils. 
GM-CSF did not induce significant G-CSF 
production, but together GM-CSF and LPS 
induced a modest level of G-CSF (Fig. 6c). 
Neither LPS nor G-CSF or GM-CSF induced 
production of GM-CSF in the cells (Fig. 6c). We 
also tested the production of CXCL-2 known to 
be induced by LPS (Fig. 6c). LPS induced a clear 
increase in CXCL-2, while G-CSF or GM-CSF 
did not stimulate CXCL2 production. 
Interestingly, in the presence of GM-CSF, the 
LPS-induced CXCL2 production doubled. Pre-
stimulating BM-derived dendritic cells with G-
CSF prior to LPS stimulation resulted in reduced 

TNF induction while the production of IL-10 was 
increased (Fig. 6d).  
  
Together, these results demonstrate that the 
presence of LPS influences the G-CSF and GM-
CSF-induced development of myeloid cells. 
Moreover the production of G-CSF depends on 
the presence of G-CSF and is enhanced by LPS 



 
 
 
 

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168 

 

and affects the inflammatory response through 
its anti-inflammatory effect on monocyte-derived 
cells. 
 

4. DISCUSSION 
 
It is well-established that a constant or regular 
intake of an energy surplus in the form of fat may 
lead to overweight or obesity and in turn 
systemic low-grade inflammation, which includes 
increased numbers of circulating neutrophils and 
monocytes [1]. However, how such diets 
influence the hematopoiesis over shorter periods 

in young and normal weight individuals has only 
been sparsely studied. In the present study we 
found that in young mice, a moderate high-fat 
(45%) diet induced increased weight after 4-5 
weeks of HFD feeding.  This was preceded by a 
prompt and transient increase in circulatory G-
CSF, neutrophils and monocytes, in G-CSF 
production by VAT and in increased production 
of neutrophils and monocytes in bone marrow.  
Of note, at the time where weight                                  
gain became significant, no signs of elevated 
circulatory neutrophils were evident in the             
mice. 

 

 
 

Fig. 3. High fat feeding induces an early transient up-regulation of neutrophils and monocytes 
in spleen. A: Gating strategy for identification of neutrophils and monocytes in spleen. The 

proportion of B: neutrophils (Ly6C
+
Ly6G

+
CD11b

+
) and C: monocytes (Ly6C

+
Ly6G

-

CD11b
+
CD115

+
) in spleen from HFD fed mice and control mice. D: Expression of genes at 

different time points in the HFD fed group compared to control mice by unpaired t-tests on dCt 
values. Mean+/-SEM, **p<0.01, * p<0.05 



 
 
 
 

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169 

 

 
 

Fig. 4. HFD induces a prompt but transient increase in the production of G-CSF from adipose 
tissue.  The concentration of A: G-CSF and B: GM-CSF in supernatant from cultured isolated 
VAT as measured by ELISA. Concentration in the HFD fed group compared to the average of 

all control mice by One-way ANOVAs. Mean+/-SEM, * p<0.05 
 

 
 

Fig. 5. HFD increases simultaneous expression of pro-inflammatory and anti-inflammatory 
genes in adipose tissue. Expression of genes in VAT at different time points in the HFD fed 

group compared to the average of all control mice by One-way ANOVA on dCt values. Mean+/-
SEM, ***<0.001, **p<0.01, * p<0.05 



 
 
 
 

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170 

 

 
 

Fig. 6. LPS enhances the G–CSF and GM-CSF-induced G-CSF, which holds anti-inflammatory 
properties. The number of A: neutrophils (Ly6G

+
Ly6C

+
CD11b

+
) and B: monocytes (Ly6C

+
Ly6G

-

CD11b
+
CD115

+
) after stimulation with LPS alone or together with G-CSF or GM-CSF of bone 

marrow for 1 or 4 days relative to the number measured in unstimulated bone marrow cells on 
Day 1. C: The concentration of G-CSF, GM-CSF and CXCL-2 in the supernatant of bone marrow 

cells after stimulation with LPS alone or together with G-CSF or GM-CSF for 1 or 4 days. D: 
The production of TNFα and IL-10 in LPS stimulated dendritic cells +/-preincubation with G-

CSF 
A transient increase in circulating neutrophils has 
been reported previously in mice fed a high-fat 

(60%) diet [15, 27]. Our data shows that also a 
considerably lower fat content in the diet (45%) 



 
 
 
 

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171 

 

may lead to this transient increase in neutrophils 
and further makes it probable that an elevated 
transient level of circulating G-CSF preceding the 
increase in neutrophils is the cause. G-CSF is 
the prime signaling molecule for mobilizing 
neutrophilic granulocytes from the bone marrow 
into circulation [28]. The early and transient up-
regulation of G-CSF in blood coincided with the 
transient increase in granulocytes measured in 
blood and in spleen during the first week of HFD 
feeding and may explain the increased level of 
neutrophils in circulation. Like others, [27]

 
we did 

not find any changes in the proportions of 
myeloid progenitor cells upon HFD feeding. This 
is in contrast to Nagareddy et al. (2014) and 
Singer et al. (2014) who found increased 
numbers of myeloid progenitors concomitantly 
with increased number of neutrophils and 
monocyte in bone marrow of obese mice [14, 
15]. Of note, these studies investigated either 
obese mice or ob/ob mice, where the              
increased number of circulating neutrophils               
and monocytes seem to be caused by                                           
metabolic conditions rather than a change in           
diet able to increase the endotoxin                      
absorption.  
 
The cause of the increased blood G-CSF 
concentration was not established. However, one 
possibility is that the high content of dietary fat in 
the gut leads to an increased lymphatic flow, in 
turn leading to an increased influx of LPS from 
the intestinal lumen through the lymphatic 
system. An increased lipid content in the diet is 
reported to increase the lymphatic flow [29] and 
may also increase the absorption of LPS [1, 30]. 
LPS may stimulate an increased production of G-
CSF in various tissue [31] in turn leading to 
recruitment of granulocytes into circulation. We 
demonstrated here that VAT from mice fed HFD 
for 3-8 days had an increased production of G-
CSF while at later time points, the production 
was reduced to the same level as VAT from mice 
on a standard diet. Other tissues than VAT may 
also induce increased production of G-CSF and 
thus contribute to a transiently increased G-CSF 
level in the blood. The most potent G-CSF-
producing cells comprise monocytes and 
macrophages, but G-CSF is also produced by 
fibroblasts and endothelial cells [32], all being 
ubiquitously present in the various tissues of the 
body. 
 
In the adipose tissue, we found a transient 
increase in the expression of Hp and Stfa2l1 
during the first week of HFD feeding indicating an 
increased number of neutrophils in the last 

differentiation stages [23]. This corresponds to 
the findings in earlier studies showing an early 
transient influx of neutrophils upon feeding a 
HFD [17, 27]. After this initial stage and after the 
G-CSF peak, we found a down-regulation of Tlr4 
expression in the HFD fed group at the time 
where mice on HFD had increased their weight 
compared to control mice, which might indicate a 
state of LPS tolerance, however such direct 
relation is purely speculative. 
 
Apart from playing a key role in the mobilization 
of neutrophils from the bone marrow into 
circulation, G-CSF is important in the promotion 
of the granulocytic lineage, mainly neutrophils 
[33]. We found an increase in both neutrophils 
and monocytes in BM from HFD fed mice during 
the first week(s), and speculated that the 
increased G-CSF could stimulate the promotion 
of these cells. To investigate this, we stimulated 
freshly isolated BM cells with G-CSF and GM-
CSF, alone or together with LPS, and found that 
in contrast to G-CSF and GM-CSF, LPS was a 
poor stimulator of neutrophils and monocytes; 
rather it seemed to halt the generation of 
neutrophils and had only modest effect on the 
generation of monocytes. Of note, the addition of 
G-CSF induced significant production of G-CSF 
in the bone marrow cells. LPS alone did not 
induce G-CSF production but slightly enhanced 
the production induced by G-CSF. Isolated BM 
cells are primarily comprised by hematopoietic 
cells but the presence of some endothelial cells 
cannot be excluded. The endothelial cells were 
previously identified as the only cells in BM 
producing G-CSF in response to LPS [7].

 
This is 

in agreement with our data showing only modest 
effect of LPS on G-CSF production. However, 
the epithelial cell-produced G-CSF might induce 
G-CSF production in the myeloid cells thus 
leading to an enhanced effect and probably 
increased generation of neutrophils and 
monocytes as indicated from our results. In 
contrast to G-CSF, GM-CSF did not stimulate G-
CSF production but together with LPS, the 
induction of both G-CSF and CXCL2 was 
enhanced. This illustrates the difference between 
the two growth factors; while GM-CSF is purely 
pro-inflammatory, G-CSF holds pro-inflammatory 
(through its neutrophil mobilizing and promoting 
property) as well as immune-regulating 
properties [34].

  

 
The increased production of G-CSF readily 
ceased within the first week of HFD feeding 
indicating the action of immune-regulating 
properties. We did not demonstrate direct 



 
 
 
 

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172 

 

causality between the G-CSF and immune 
regulation in vivo, but we could show that G-CSF 

inhibited TNF production while increasing IL-10 
production in vitro. Especially monocytes and 
their derivatives expressing the G-CSF receptor 
are influenced by G-CSF in an anti-inflammatory 
way leading to attenuated production of pro-
inflammatory cytokines upon LPS stimulation 
[35]. Ligation of the G-CSF receptor induces 
internalization and degradation of the receptor 
resulting in down-regulation of the 
responsiveness to G-CSF. Mice challenged with 
LPS after preteatment with G-CSF, were 
protected against the dose of LPS that killed non-
pretreated mice through a mechanism involving 
inhibition of TNFα [36]. Blood taken from healthy 
humans injected with G-CSF responded with less 
cytokine production when stimulated in vitro with 
LPS [37] and volunteers pretreated with G-CSF 
before injection of minute doses of LPS exhibited 
lower levels of the pro-inflammatory cytokines IL-

6 and TNF [38].
 
Together, this points towards a 

strong anti-inflammatory activity of G-CSF, 
however, for how long the elevated serum G-
CSF found in mice fed a HFD may prevent 
inflammation remains to be investigated as does 
the possibility of other mechanisms taking over in 
preventing HFD-induced inflammatory 
responses. To this end, IL-10 produced by 
monocytes and macrophages holds even 
stronger anti-inflammatory activity and influences 
monocytes as well as adipocytes [39, 40] and IL-
10 from monocytes was recently demonstrated to 
induce epigenetic chances in adipocytes [40]. 
Whether IL-10 is involved in mechanisms 
causing establishment of long-term endotoxin 
tolerance is however purely speculative and the 
presented data calls for new studies that 
investigate a possible long term anti-
inflammatory effect of G-CSF. 

 
5. CONCLUSION 
 
In conclusion, the presented data confirm 
previous reports showing that HFD fed to mice 
induces a transient increase in circulatory 
neutrophils and monocytes. This is paralleled 
with a transient peak in G-CSF. The VAT 
contributes to the transient production of G-CSF, 
which is followed by down-regulation of TLR4 
expression. As G-CSF inhibits production of 

TNF and up-regulates IL-10 production we 
suggest that the transient G-CSF peak stimulates 
endotoxin tolerance. This may confer an anti-
inflammatory state to the body upon high-fat 
meals that may influence post-prandial 
responses and might contribute to the high 

variations seen in the inflammatory response to a 
single meal. Further studies are                            
warranted to pursue the possible importance of 
G-CSF in post-prandial inflammatory             
response. 
 

DISCLAIMER 
 

The products used for this research are 
commonly and predominantly use products in our 
area of research and country. There is absolutely 
no conflict of interest between the authors and 
producers of the products because we do not 
intend to use these products as an avenue for 
any litigation but for the advancement of 
knowledge. Also, the research was not                
funded by the producing company rather                                  
it was funded by personal efforts of the                
authors. 
 
SUPPLEMENTARY MATERIALS 
 

Supplementary materials available in this link: 
https://www.journalaji.com/index.php/AJI/libraryFi
les/downloadPublic/3 
 

COMPETING INTERESTS 
 

Authors have declared that no competing 
interests exist. 
 

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