Pre-stimulation of Bone-marrow Derived Eosinophils with CCL24

Alters Responses to TLR Ligands and Helminth Extracts

B. C. Buerfent

, A. Ehrens

, W. Stammin

, A. Hoerauf

1,2

, M. P. Hübne

1,*

Institute for Medical Microbiology, Immunology and Parasitology, University Hospital of Bonn, Germany

German Center for Infection Research (DZIF), partner site Bonn-Cologne, Bonn, Germany

Keywords: Eosinophils, Filaria, Helminth, TLR, LPS, Eotaxin

Abstract: Eosinophil granulocytes are a hallmark of helminth infection, provide protection against helminth infections

and elicit detrimental effects during allergy and asthma. Moreover, eosinophils are associated with diabetes,

arthritis and sepsis. Thus, eosinophils have a broad range of implications, contributing to homeostasis as

well as pathogenesis of various diseases. In the current study we used murine bone-marrow derived

eosinophils (bmEos) to investigate the impact of eosinophil pre-stimulation with the chemoattractants

CCL11 and CCL24 (eotaxin-1 and 2) on TLR2, TLR4 and filarial extract-induced eosinophil responses.

Generation of bmEos consistently resulted in approximately 50 million bmEos from a single donor mouse

and a purity and viability above 95%. Upon stimulation with CCL24, TLR2, TLR4, and filarial extract,

bmEos released different quantities of IL-4, IL-6, CCL5, as well as CXCL1. CCL24 pre-stimulation

partially affected those responses. Furthermore, CCL24 pre-stimulation of bmEos reduced the expression of

the eotaxin receptor CCR3 independently of TLR2 stimulation. In contrast, expression of adhesion molecule

ICAM-1 was increased by TLR2 stimulation, but not affected by CCL24 pre-stimulation. Hence, our results

reveal an impact of CCL24 on bmEos activation. bmEos present a promising tool to study eosinophil

responses that may help to further characterize their role in different immunological contexts and overcome

the limitations given by the low eosinophil frequencies present in non-helminth-infected individuals.

1 INTRODUCTION

Eosinophil granulocytes are most famous for their

involvement in the pathogenesis of allergies and

asthma (Fulkerson and Rothenberg, 2013) as well as

their characteristic expansion and protective effect

during helminth infection (Gentil et al., 2014).

However, eosinophils further support anti-bacterial

responses, contribute to metabolic homeostasis and

impact autoimmune diseases. Accordingly,

eosinophils recognize pathogen associated molecular

patterns and possess anti-bacterial functions due to

the release of bactericidal NET like structures and

phagocyte-recruiting chemokines and are discussed

as potential marker for the severity of bacterial

sepsis (Merino et al., 2012). Moreover, adipose

tissue eosinophils help to maintain glucose and

insulin tolerance by driving alternative macrophage

activation via the release of IL-4 (Wu et al., 2011).

Such a beneficial role of eosinophils was also

described during inflammatory arthritis, which was

mitigated by helminth-induced eosinophils (Chen et

al., 2016).

Eosinophil granulocytes produce and detect

numerous chemokines and cytokines and express

pattern recognition receptors including toll-like-

receptors (Rosenberg et al., 2013). In general, IL-5

is the main inducer of eosinophils and eotaxins that

bind to the chemokine receptor CCR3 direct

eosinophils to the site of inflammation. Thus,

eosinophils are involved in a broad range of

homeostatic and inflammatory conditions and

essentially modulate immune responses and

pathogenesis. We here provide evidence for the

impact of the eotaxins CCL11 and CCL24 on

subsequent TLR-induced and filarial extract-induced

immune responses of bone-marrow derived

eosinophils (bmEos), which may contribute to the

diverse spectrum of eosinophil functions.

Buerfent, B., Ehrens, A., Stamminger, W., Hoerauf, A. and Hübner, M.

Pre-stimulation of Bone-marrow Derived Eosinophils with CCL24 Alters Responses to TLR Ligands and Helminth Extracts.

DOI: 10.5220/0008787800050009

In Proceedings of the 2nd Syiah Kuala International Conference on Medicine and Health Sciences (SKIC-MHS 2018), pages 5-9

ISBN: 978-989-758-438-1

 2020 by SCITEPRESS – Science and Technology Publications, Lda. All rights reser ved

2 METHODS

2.1 Ethics Statement and Mice

BALB/c mice (Janvier, Saint Berthevin Cedex,

France) were housed at the Institute for Medical

Microbiology, Immunology and Parasitology of the

University Hospital Bonn, Germany, with access to

food and water ad libitum. All experiments were

approved by the Landesamt für Natur, Umwelt und

Verbraucherschutz, Cologne, Germany and

performed according to the European Union animal

welfare guidelines.

2.2 In Vitro Eosinophil Differentiation

from Bone-marrow

Eosinophils were differentiated from bone-marrow

of naïve adult mice by stimulation with 100ng/ml

stem cell factor (SCF) and FMS-like tyrosine kinase

3 ligand (FLT3L) for four days followed by culture

with 20ng/ml IL-5 for eight days (all Peprotech,

Rocky Hill, USA, Fig. 1A) (Dyer et al., 2008). Cells

were adjusted to 1x10

cells/mL and incubated at

37°C and 5% CO

. Half of the medium containing

advanced RPMI 1640, 20% heat-inactivated fetal

calf serum, 1M HEPES, 10.000 IU/mL penicillin,

10µg/mL streptomycin, 1X GlutaMAX

(all

Gibco® Technologies, Waltham, USA) was

replaced every other day. Adherent cells were

removed at day 8 and eosinophil purity was checked

at day 12.

2.3 In Vitro Stimulation of

Bone-marrow Derived Eosinophils

1x10

bmEos were pre-stimulated for 24 hours with

100ng/mL CCL11 or CCL24 (both Peprotech,

Rocky Hill, USA) in eosinophil growth medium.

Subsequently, cells were re-stimulated for 24 h with

200ng/mL lipopolysaccharide (LPS) ultrapure,

500ng/mL Pam3CSK4 (P3C) (both InvivoGen, San

Diego, USA) or 25µg/mL Litomosoides sigmodontis

crude adult worm extract (LsAg). LsAg was

prepared as previously described (Gentil et al.,

2014).

2.4 Flow Cytometry, Fluorescence

Microscopy and Enzyme Linked

Immunosorbent Assay

After blocking with PBS containing 1% bovine

serum albumin and 0.1% rat IgG (Sigma-Aldrich, St.

Louis, USA) for 30 min, bmEos were washed and

stained with combinations of anti-SiglecF AL647

(BD Pharmingen, San Diego, USA), anti-

CD54/ICAM-1 AL488, and anti-CD193/CCR3 PE

(both BioLegend, San Diego, USA). Data were

acquired using a BD FACS Canto (BD Bioscience,

San Jose, USA) and analyzed by FlowJo v10

software (Tree Star, Ashland, USA).

For confocal fluorescence microscopy bmEos

were fixed with 3% formaldehyde fixative solution

for 20 min on 15 mm glass slides (P+W

Medizintechnik, Berlin, Germany) and stained with

rabbit anti- eosinophil cationic protein (ECP)

(Biorbyt Ltd, Cambridge, UK) for 1 h followed by 1

h staining with goat anti-rabbit FITC (Invitrogen,

Waltham, USA) and anti-SiglecF AL647. DAPI was

stained for 10 min (Sigma-Aldrich, Steinheim,

Germany). Z-stack pictures were taken with the

Zeiss LSM 710 and the ZEN 2.3 software (both Carl

Zeiss AG, Oberkochen, Germany).

Cytokine and chemokine concentrations were

determined from supernatants by ELISA according

to kit protocols (IL-6 and TNFα (eBioscience);

CXCL1 and CCL5 (R&D, Minneapolis, USA) using

a SpectraMAX 190 system and SoftMax Pro 6.5

software (Molecular Devices, Sunnyvale, USA).

2.5 Statistical Analysis

Statistical analysis was performed using Prism

GraphPad 5.01 (GraphPad Software, San Diego,

USA). Statistical significance was tested by Kruskal-

Wallis test followed by Dunn's Multiple Comparison

post hoc test. Significance is defined as p value <

0.05 and error bars represent means ± SEM.

3 RESULTS

3.1 Generation of Murine Bone-marrow

Derived Eosinophils

Flow cytometric analysis of in vitro generated

bmEos revealed a 98% purity of SiglecF

CCR3

cells (Fig. 1A, B). H&E staining as well as

fluorescence microscopy using anti-ECP, anti-

SiglecF and DAPI confirmed that bmEos had the

typical eosinophil appearance with eosin-stained

granule, U-shaped nucleus and contained ECP (Fig.

1C, D). The viability of bmEos was analyzed by

Annexin V and propidium iodide staining after

twelve days of culture and was consistently above

95% (Fig. 1E). In general, 50-80 million bmEos

SKIC-MHS 2018 - The 2nd Syiah Kuala International Conference on Medicine and Health Sciences

were obtained from one single donor mouse (data

not shown).

Figure 1: In vitro differentiation of bone-marrow derived

eosinophil granulocytes. Bone-marrow from tibiae and

femur of 6 week-old BALB/c mice were stimulated with

100ng/ml recombinant mouse SCF and recombinant

mouse FLT3L for four days followed by eight day

stimulation with recombinant IL-5 (A). Analysis of the

purity of SiglecF+CCR3+ eosinophils by flow cytometry

on day 12 (B). Fluorescence microscopy of eosinophils

stained with anti-SiglecF (red), anti-ECP (green) and

DAPI (blue) (C) and H&E staining of differentiated

eosinophils (D). Viability of differentiated eosinophils as

determined by Annexin V and propidium iodide staining

via flow cytometry (E).

3.2 CCL24 Modulates Cytokine and

Chemokine Release by Bone-marrow

Derived Eosinophils

Since eosinophils are predominantly recruited by the

chemokines CCL11 and CCL24, we investigated

their role on bmEos activation in vitro. BmEos were

stimulated for 24h with the filarial extract LsAg, the

TLR4 agonist LPS and the TLR1/2 agonist P3C, in

the presence or absence of CCL11 or CCL24 pre-

stimulation. IL-4 release by bmEos was not induced

by CCL11, LPS, P3C or LsAg stimulation alone, but

tended to be increased upon stimulation with

CCL24. Pre-stimulation of bmEos with CCL24

before LPS and P3C re-stimulation resulted in a

significantly increased release of IL-4 compared to

LPS- and P3C-only stimulated controls. Similarly,

CCL24 pre-stimulation significantly increased

LsAg-induced IL-4 release compared to

unstimulated controls (p<0.05). LPS and P3C

potently induced IL-6 and CCL5/RANTES by

bmEos (Fig. 2B, C). While pre-stimulation with

CCL11 had no impact on subsequent LsAg-, LPS- or

P3C-induced IL-6, CCL5 and CXCL1 release by

bmEos, CCL24 pre-stimulation reduced P3C-

stimulated CCL5 production and increased by trend

LsAg-induced IL-6 release (Fig. 2B, C). CXCL1

release was significantly induced by P3C but none

of the other stimulations alone (Fig. 2D). However,

pre-stimulation with CCL24 led to a significantly

increased CXCL1 release upon LPS re-stimulation

(Fig. 2D). Those results indicate that CCL24 pre-

stimulation affects bmEos responses to subsequent

stimuli.

Figure 2: CCL24 modulates cytokine and chemokine

release by bone-marrow derived eosinophils.

Concentrations of IL-4 (A), IL-6 (B), RANTES/CCL5 (C),

and KC/CXCL1 (D) in the supernatant after a total of 48h

stimulation. Cells were untreated or pre-stimulated with

CCL11 or CCL24 for 24 hours followed by re-stimulation

with LPS, Pam3CSK4 (P3C) or crude Litosomoides

sigmodontis adult worm extract (LsAg) for additional 24

hours. Data of two independent and pooled in vitro

experiments with 7 replicates are shown. Data is presented

as mean + SEM and analyzed for statistical significance

using Kruskal-Wallis test followed by Dunn’s posthoc test

(*p<0.05, **p<0.01, ***p<0.01).

3.3 CCL24 Pre-Stimulation Reduces

the Expression of CCR3

Since CCL24 pre-stimulation and P3C

stimulation induced bmEos activation, the

impact of CCL24 pre-stimulation on the

expression of CCR3 and ICAM-1 were

investigated. The expression of CCR3

significantly decreased upon CCL24 pre-

stimulation and was not altered by TLR2

stimulation (Fig. 3A, B). In contrast, ICAM-1

expression was increased by P3C stimulation,

but not altered by CCL24 pre-stimulation (Fig.

3C, D). These results indicate that bmEos react

upon TLR activation with an increased ICAM1

expression, which may facilitate their tissue

migration and reduce the expression of the CCR

for the major eosinophil recruiting factors after

pre-stimulation with CCL24.

Pre-stimulation of Bone-marrow Derived Eosinophils with CCL24 Alters Responses to TLR Ligands and Helminth Extracts

Figure 3: Treatment with CCL24 modulates CCR3

expression. Bone-marrow derived eosinophils were pre-

stimulated with CCL24 for 24h followed by six hours re-

stimulation with Pam3CSK4 (P3C). Histograms and MFI

of CCR3 (A, B) and CD54/ICAM-1 (C, D) SiglecF

eosinophils are shown. Data of two independent and

pooled in vitro experiments with 6 replicates are shown

and are presented as mean + SEM and analyzed for

statistical significance using Kruskal-Wallis followed by

Dunn’s post hoc test (*p<0.05, **p<0.01, ***p<0.01).

4 DISCUSSION AND

CONCLUSION

In this study we describe the in vitro generation

of bmEos and the impact of bmEos pre-

stimulation with CCL11/CCL24 on cytokine

and chemokine release in response to TLR

ligands and LsAg. The stimuli chosen for this

study induced different cytokine/chemokine

pattern from bmEos, with P3C and LPS

triggering CCL5 and IL-6 release, P3C

inducing CXCL1 production and CCL24 the

release of IL-4. Interestingly, LsAg-induced

cytokine/chemokine release by bmEos was only

present after pre-stimulation with CCL24,

resulting in increased IL-6 and IL-4 release.

CCL24 pre-stimulation also increased IL-4

responses after re-stimulation with P3C and

LPS. Such an effect by eotaxin to induce the

release of preformed IL-4 was also observed for

human eosinophils that was additionally

enhanced by IL-5 (Bandeira-Melo et al., 2001).

This indicates that in the context of increased

CCL24 concentrations, as they may occur

during type 2 inducing helminth infections,

eosinophils may be more prone to support type

2 immune responses independent on the

stimulus, which may render them more efficient

for protection against filarial infections (Gentil

et al., 2014). However, pre-stimulation with

CCL24 also triggered the release of pro-

inflammatory mediators like CXCL1 upon LPS

re-stimulation and IL-6 after LsAg re-

stimulation and bmEos responded to TLR2 and

TLR4 stimuli by the release of IL-6. Those

results suggest that bmEos may also support

anti-bacterial responses by triggering neutrophil

recruitment via CXCL1 and acute phase

responses, which can be in part enhanced by

CCL24 pre-stimulation. BmEos further reacted

upon TLR2 activation with an increased

ICAM1 expression, which may increase cell

contact with other leucocytes and promote

inflammation (Czech et al., 1993). In contrast,

CCR3 expression of BmEos was reduced by

CCL24 treatment independently of TLR2

stimulation suggesting two independent

mechanisms of eosinophil migration and

eosinophil activation (Humbles et al., 2002). In

summary, our data demonstrate that bmEos

possess characteristics that are known from ex

vivo isolated eosinophils and indicate that

CCL24 pre-treatment modulates eosinophil

responses.

SOURCES OF FUNDING

This work was funded by the German Research

Foundation (HU 2144/1-1). BB and AE are

supported by the Jürgen Manchot Stiftung,

Düsseldorf. AH is a member of the German

Center for Infection Research (DZIF) and of the

Excellence Cluster Immunosensation (DFG,

EXC 1023).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

SKIC-MHS 2018 - The 2nd Syiah Kuala International Conference on Medicine and Health Sciences

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Pre-stimulation of Bone-marrow Derived Eosinophils with CCL24 Alters Responses to TLR Ligands and Helminth Extracts