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Growth Hormone Aggregates Activation of Human Dendritic Cells Is Controlled by Rac1 and PI3 Kinase Signaling Pathways
Journal Pre-proof Growth hormone aggregates activation of human dendritic cells is controlled by Rac1 and PI3 kinase signaling pathways Myriam Nabhan, Yann Gallais, Marc Pallardy, Isabelle Turbica PII:
S0022-3549(19)30577-5
DOI:
https://doi.org/10.1016/j.xphs.2019.09.007
Reference:
XPHS 1708
To appear in:
Journal of Pharmaceutical Sciences
Received Date: 14 June 2019 Revised Date:
30 August 2019
Accepted Date: 6 September 2019
Please cite this article as: Nabhan M, Gallais Y, Pallardy M, Turbica I, Growth hormone aggregates activation of human dendritic cells is controlled by Rac1 and PI3 kinase signaling pathways, Journal of Pharmaceutical Sciences (2019), doi: https://doi.org/10.1016/j.xphs.2019.09.007. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
1
Growth hormone aggregates activation of human dendritic cells is controlled by Rac1 and PI3
2
kinase signaling pathways
3
Myriam Nabhan , Yann Gallais , Marc Pallardy and Isabelle Turbica
4
1 Inflammation Chimiokines et Immunopathologie, INSERM, Fac. de pharmacie - Univ.Paris-Sud,
5
Université Paris-Saclay, Châtenay-Malabry, France.
6
2 Formerly: Inflammation Chimiokines et Immunopathologie, INSERM, Fac. de pharmacie - Univ.Paris-
7
Sud, Université Paris-Saclay, Châtenay-Malabry, France.
8
Current address: CEA-Saclay, Institute Frederic Joliot, Service d'Ingénierie Moléculaire des Protéines,
9
Gif-sur-Yvette, France
1,§
2,§
1
1*
10 11
§: Both authors contributed equally to this work.
12 13
* Corresponding Author: [email protected]; +33(0)146835299
14
KEYWORDS: protein aggregation, immunogenicity, immune response, in vitro model(s), cell biology
15
SHORT TITLE: CXCL10 regulation by growth hormone aggregates
1
16
ABSTRACT
17
The presence of protein aggregates in biological products is suggested to promote immunogenicity,
18
leading to the production of anti-drug antibodies with neutralizing capacities. This suggests a CD4 T-cell
19
dependent adaptive immune response thus a pivotal role for antigen presenting cells, such as dendritic
20
cells (DC). We previously showed that human growth hormone (hGH) aggregates induced DC maturation,
21
with notably an increase in CXCL10 production. DC phenotypic modifications were sufficient to promote
22
allogeneic CD4+ T-cell proliferation with Th1 polarization. In this work, we identified the main intracellular
23
signaling pathways involved in DC activation by hGH aggregates, showing that aggregates induced p38
24
MAPK, ERK and JNK phosphorylation, as well as NF-κB subunit p65 nuclear translocation. Next,
25
investigating the implication of Rho GTPases and phosphoinositide 3-kinase (PI3K) in activated DC
26
showed that Rac1 and Cdc42 regulated the phosphorylation of MAP kinases, whereas PI3K was only
27
implicated in JNK phosphorylation. Furthermore, we showed that Rac1 and PI3K pathways, but not
28
Cdc42, regulated the production of CXCL10 via the MAP kinases and NF-κB. Taken together our results
29
bring new insight on how protein aggregates could induce DC activation, leading to a better understanding
30
of aggregates role in therapeutic proteins immunogenicity.
+
31 32
Abbreviations: DC: dendritic cells; ERK: extracellular signal-regulated kinase; hGH: human growth
33
hormone; JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; moDC: monocyte-
34
derived dendritic cells; NF-κB: nuclear factor κB ; PI3K: phosphoinositide-3 kinase; Th: T helper.
2
35
INTRODUCTION
36
Patients treated with biological products (BP) frequently develop anti-drug antibodies (ADA) with
37
potentially neutralizing capacities leading to loss of clinical response
38
BP have been suggested as one of the factors promoting immunogenicity, as their occurrence seems to
39
be correlated with ADA development in mice models
40
dependent adaptive immune response and therefore a pivotal role for antigen presenting cells, such as
41
dendritic cells (DC) 3. We, and others, have previously shown that protein aggregates could act as a danger
42
signal and be recognized by dendritic cells inducing their maturation and CD4 T-cell response
43
previous work, we demonstrated that hGH aggregates were able to induce monocyte-derived DC (moDC)
44
maturation, showed by a significant increase of surface markers expression and a high production of pro-
45
inflammatory cytokines and inflammatory chemokines with notably an increase in CXCL10 secretion
46
compared to the native protein. Moreover, moDC phenotype induced by the hGH aggregates was able to
47
drive allogeneic CD4 T cells towards a T helper 1 (Th1) profile. In this previous work, aggregates were
48
generated by submitting native human growth hormone (hGH, Somatropin) to a stir stress .
49
CXCL10, also called IP-10 (interferon γ-induced protein 10), is an inflammatory chemokine that binds to
50
CXCR3, a receptor expressed on a wide range of immune cells including T lymphocytes, natural killer
51
cells (NK), DC and macrophages, thus favoring their activation and recruitment
52
upregulates T-bet expression as well as IFN-γ (interferon-γ) production in CD4 T cells thus promoting
53
Th1 polarization . The secretion of CXCL10 in response to external stimuli is dependent on an array of
54
transcription factors including NF-κB (nuclear factor κB), AP-1 (activator protein 1) and ISRE (interferon-
55
stimulated response element)
56
pathways that could be involved in particles internalization and actin cytoskeleton reorganization
57
Following these observations, we raised the hypothesis that protein aggregates could trigger specific
58
signaling pathways in human dendritic cells. hGH aggregates could also act differently than aggregates
59
from therapeutic antibodies usually used as a model. In the present work, we searched for pathways that
60
could be rapidly activated by these aggregates. CXCL10 is an important chemokine for T-cell biology and
61
in our hand it is produced by moDC treated with hGH aggregates but not with therapeutic antibody
62
aggregates
1,2
. The presence of aggregates in
3,4
+
. Production of ADA suggests a CD4 T-cell
+
5,6
. In a
+
5
7,8
. Moreover, CXCL10
+
9
10
. These transcription factors are regulated by upstream signaling 11
.
12
. CXCL10 can then be used as a specific biomarker of DC activation by GH-type
3
63
aggregates. For the first time, our results highlighted the involvement of small GTPases and of
64
phosphoinositide-3 kinase (PI3K) in the regulation of the signaling pathways induced by aggregates in
65
moDC.
4
66
MATERIAL AND METHODS
67
Samples preparation
68
Lyophilized human Growth Hormone (hGH) was purchased from European Pharmacopoeia (ref:
69
S0947000; EDQM Council of Europe, Strasbourg, France) and diluted to a final concentration of 1 mg mL
70
1
71
samples were stirred at 1000 rpm for 72 hours at room temperature in 5 mL glass tubes with an 8x2 mm
72
Teflon stirrer bar. Endotoxin was quantified for native and aggregated hGH by using a colorimetric assay
73
(ToxinSensor, Genscript, Paris, France), according to the manufacturer’s instructions.
74
Generation of human monocyte-derived dendritic cells (moDC)
75
Peripheral blood mononuclear cells (PBMC) were purified from buffy coats obtained from Etablissement
76
Français du Sang (EFS Rennes, France) by density centrifugation on a Ficoll gradient (lymphocyte
77
separation medium; GE Healthcare, Buc, France). Healthy donors gave their written consent for the use
78
of blood donation for research purposes. MoDC preparation was previously described .
79
Treatment of moDC
80
After differentiation, moDC were stimulated with 50 µg mL
81
times. In some experiments, moDC were pretreated for 30 min with PD98059 (20 µM), SP600125 (20
82
µM) or SB203580 (20 µM) and for 1 h with Bay 11-7085 (3 µM). For Rho GTPases and PI3K
83
experiments, moDC were pretreated for 1h with Ly294002 (25 µM), Rac1 inhibitor (50 µM) or ML141 (10
84
µM). All inhibitors (diluted in DMSO) were purchased from Calbiochem, Darmstadt, Germany.
85
Quantitative real-time RT-PCR assay of CXCL10 in moDC
86
Total RNA was extracted after lysis of treated cells pretreated or not with inhibitors, using the Nucleospin
87
RNA kit (Macherey Nagel, Hoerdt, France), according to the manufacturer’s instructions. First-strand
88
cDNA was synthesized from total RNA, as previously described
89
performed by use of the SYBR Green technology on a CFX96 system (Bio-Rad Laboratories, Marnes la
90
Coquette, France), as previously described
91
CFX Manager software. Ratios were calculated as the geometrical mean of (1 + E)
92
the efficiency and ∆∆Ct is the target gene expression of treated cells compared with untreated cells,
93
corrected to the expression of the reference genes gapdh and β-actin. Results were expressed as the
-
5
with sterile PBS. Aggregates preparation by stir stress was previously described . In brief, 1 mL
5
-1
of native or aggregated hGH for indicated
13
. Real-time quantitative PCR was
13
. Quantification was performed with Bio-Rad Laboratories -∆∆Ct
values, where E is
5
-∆∆Ct
-∆∆Ct
94
fold-factor induction [ratio of (1 + E)
95
specific primers, purchased from Eurofins Genomics (Ebersberg, Germany), were used (forward and
96
reverse, respectively): cxcl10: 5’-TCT AAG TGG CAT TCA AGG AGT ACC-3’ and 5’-AAA GAC CTT GGA
97
TTA ACA GGT TGA-3’ ; gapdh: 5’-CAG CCT CAA GAT CAT CAG CA-3’ and 5’-TGT CGT CAT GAG
98
TCC TTC CA-3’; β-actin: 5’-GGC ATC CTC ACC CTG AAG TA-3’ and 5’-GCA CAC GCA GCT CAT TG
99
TAG-3’.
of treated cells/(1 + E)
of untreated cells]. The following
100
Immunoblot analysis of signaling pathways in moDC
101
After pretreatment or not with inhibitors, moDC were incubated for 30 min with native or aggregated hGH,
102
harvested and washed in cold phosphate buffered saline before lysis. 30 micrograms of denatured protein
103
were loaded onto 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes, which
104
were successively incubated with antibodies directed against the phosphorylated forms of JNK
105
(Thr183/Tyr185), p38 MAPK (Thr180/Tyr182), ERK1/2 (Thr202/Tyr204) and the NF-κB p65 subunit
106
(Ser536) and an antibody against total p38 MAPK as a loading control (Ozyme, Montigny-Le-Bretonneux,
107
France). Immunoreactive bands were detected by their chemiluminescence by use of the ChemiDoc
108
XRS+ System (Bio-Rad Laboratories, Marnes-La-Coquette, France). Bands were quantified with
109
ImageLab software. In some experiments, NF-κB activation was also evaluated by a pull-down assay, as
110
described previously
111
lysed in NP-40 lysis hypertonic buffer. Whole-cell extracts (150 µg) were incubated at 4°C for 90 min with
112
2 µg double-stranded 59-biotinylated oligonucleotide coupled to 30 mL streptavidin-agarose beads
113
(Sigma-Aldrich, St-Quentin Fallavier, France). Complexes were washed in binding buffer and eluted by
114
boiling in reducing sample buffer before protein separation by SDS-PAGE gel, followed by Western blot
115
analysis of the NF-κB p65 subunit, as described above.
116
CXCL10 quantification in moDC supernatants
117
After pretreatment or not with inhibitors, moDC were stimulated for 24 h with native or aggregated hGH.
118
Meso Scale Discovery (MSD, Rockville, Maryland, USA) U-plex assay was performed on culture
119
supernatants according to the manufacturer’s instructions to measure CXCL10 (IP-10). The quantification
120
range for CXCL10 is: 2.2–9040 pg mL .
121
Statistical analysis
14
. In brief, after a 30 min incubation with native or aggregated hGH, moDC were
-1
6
122
Data are expressed as means ± SEM. Differences between groups were evaluated with the Mann–
123
Whitney U-test (Prism software, GraphPad, La Jolla, CA). P-values below 0.05 were considered to denote
124
statistical significance.
7
125
RESULTS
126
MAPKs and NF-κB are activated in response to hGH aggregates
127
In order to study the involvement of mitogen-activated protein kinase (MAPKs) and NF-κB pathways in
128
moDC in response to aggregates, we first analyzed their phosphorylation by Western blotting. Native
129
hGH had little effect on proteins phosphorylation after 30 min of incubation compared to non-stimulated
130
cells (Fig. 1(a)). However, aggregated hGH significantly increased phosphorylation of p38 MAPK,
131
extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and NF-κB subunit p65 (Fig.
132
1(a)). Using a pull-down assay, we showed an increased NF-κB p65 DNA-binding activity after 30 min of
133
treatment with aggregated hGH compared to native hGH (Fig. 1(b)). The overall results confirmed the
134
involvement of p38 MAPK, ERK, JNK and NF- κB in the intracellular signaling mediated by hGH
135
aggregates in moDC.
136
Control of p38 MAPK, ERK & JNK activation by Rho GTPases & PI3K
137
Particles and aggregates are known to provoke cell membrane alterations leading to cytoskeleton
138
reorganization and small G proteins activation
139
MAPKs activation following hGH aggregates treatment by using pharmacological inhibitors of these small
140
G proteins (Rac1 inhibitor and ML141, a Cdc42 inhibitor). Cells were pretreated for 1 h with these
141
inhibitors and then stimulated for 30 min with hGH aggregates. We first assessed the effect of the
142
inhibitors alone. Indeed, Cdc42 inhibitor alone slightly increased the phosphorylation of ERK whereas
143
PI3K inhibitor alone slightly increased JNK phosphorylation (Supplementary Fig. S1). Then, we
144
determined the effect of hGH aggregates on MAPKs activation, by taking into account the by stander
145
effect of the inhibitors (Fig.2). We observed that p38 MAPK and ERK phosphorylation was significantly
146
decreased by the inhibitors of Cdc42 and Rac1 whereas JNK phosphorylation showed a trend to
147
decrease with Cdc42 inhibitor (Fig. 2 (a) and (b)).
148
Knowing that PI3K is also involved in controlling cytoskeletal changes upon large particles’
149
internalization , we investigated PI3K impact on the MAPKs activation in response to hGH aggregates.
150
Cells were pretreated for 1 h with a PI3K inhibitor (Ly294002) and then stimulated for 30 min with hGH
151
aggregates. We observed that p38 MAPK and ERK phosphorylation was not altered by the PI3K inhibitor.
15
. Therefore, we assessed if these proteins could regulate
15
8
152
However, JNK phosphorylation showed a trend to decrease after pretreatment with this inhibitor (Fig. 2
153
(a) and (b)).
154
hGH aggregates regulate cxcl10 mRNA expression via MAPK and NF-κB pathways
155
In order to connect the upregulation of MAPKs and NF-κB signaling pathways induced by aggregated
156
with cellular responses, we used well-described pharmacological inhibitors of these pathways and
157
investigated their implication in cxcl10 expression. At day 4, cells were pretreated for 1 h with Bay 117085
158
(a NF-kB inhibitor), or for 30 min with either PD98059 (an ERK inhibitor), SP600125 (a JNK inhibitor) and
159
SB203580 (a p38 MAPK inhibitor) and then stimulated for 4 h with hGH aggregates. Inhibitors
160
concentrations and treatment timelines were optimized to avoid cellular toxicity, according to previous
161
studies
162
inhibitors, suggesting that hGH aggregates modulate cxcl10 expression via NF-κB, ERK, JNK and p38
163
MAPK signaling pathways (Fig. 3(a)).
164
Rac1 and PI3K control hGH aggregates-mediated production of CXCL10
165
Next, using the pharmacological inhibitors of Rho GTPases and PI3K, we analyzed the impact of Rac1,
166
Cdc42 and PI3K on CXCL10 secretion in aggregates-treated moDC. Cells were pretreated for 1 h with
167
inhibitors and then stimulated for 24 hours with hGH aggregates. We observed that PI3K and Rac1
168
inhibitors downregulated CXCL10 production, whereas Cdc42 inhibitor had little effect on CXCL10
169
secretion (Fig. 3(b)). These results suggest that PI3K and Rac1 are involved in DC activation induced by
170
hGH aggregates
16
. Results showed that cxcl10 expression was decreased when moDC were pretreated with these
9
171
DISCUSSION
172
Aggregation of therapeutic proteins has been associated with an increased susceptibility to develop an
173
immune response targeting the BP
174
immune danger signal and initiate a protein-specific adaptive immune response, we analyzed the impact
175
of hGH aggregates on the regulation of the intracellular mechanisms leading to moDC maturation. We
176
had previously shown that hGH aggregates, generated under stir stress, induced moDC maturation,
177
increasing the expression of the surface markers CD80, CD83, CD86 and HLA-DR as well as the
178
secretion of the cytokines IL-6, IL-8, IL-12p40, and the chemokines CCL2, CCL3, CCL4 and CXCL10 . In
179
the present study, we confirmed this inflammatory phenotype and used the production of the inflammatory
180
chemokine CXCL10 as a biomarker of moDC activation in response to hGH aggregates. The choice to
181
focus on CXCL10 was made for two main reasons: first, it is an important chemokine playing a central
182
role in T-helper cell polarization and second, we described its production in response to hGH aggregates
183
but not with therapeutic antibody aggregates
184
To study intracellular mechanisms regulated in aggregates-stimulated moDC, we first showed that
185
aggregates increased phosphorylation of p38 MAPK, ERK, JNK as well as NF-κB subunit p65 binding.
186
This observation seems in agreement with a recent study by Polumuri et al. also showing the implication
187
of intravenous immunoglobulin (IVIG) and bevacizumab aggregates (shaken or stirred) in the activation of
188
MAPKs in human peripheral blood mononuclear cells
189
phosphorylation, but not p38 or JNK, was induced in moDC in response to infliximab aggregates
190
Previous reports also demonstrated activation of MAPKs and NF-κB subunit p65 in moDC in response to
191
danger signals such as lipopolysaccharide (LPS) and contact sensitizers, such as nickel
192
then interested in the upstream signaling pathways that participated in DC response to hGH aggregates.
193
Both Rac1 and PI3K are highly involved in actin remodeling observed in phagocytosis, the internalization
194
mechanism of large insoluble particles by innate immune cells such as macrophages, neutrophils and
195
dendritic cells
196
to hGH aggregates and these aggregates, having a diameter larger than 0.5 µm , induced cytoskeletal
197
changes leading to their possible internalization via phagocytosis. A previous report on sub-visible
198
aggregates effect on moDC showed that stir-stressed rituximab was efficiently uptaken by moDC and
3,17
. In order to further understand how protein aggregates act as an
5
12
.
18
. Furthermore, we recently showed that ERK 12
.
14,16,19
. We were
11
. Our observations suggest that Rac1 and PI3K are implicated in the response of moDC 5
10
20
199
rapidly accumulated in late endosomes
200
aggregates recognition and internalization mechanisms by DC. The involvement of small G protein
201
signalization suggests that the cell membrane could somehow be affected by GH particulates. Therefore,
202
it is possible that GH aggregates could follow different pathways than those described for antibodies
203
aggregates with involvement of Toll-like receptors and Fc receptors . However, it is unlikely that GH
204
aggregates bind to GH receptors expressed on moDC since aggregates formation often requires
205
conformational misfolding of the monomers to favor interactions. Indeed, we previously showed that GH
206
aggregates formation induced a loss of α-helix in secondary structure .
207
In order to link the intracellular events upregulated by hGH aggregates with the inflammatory moDC
208
phenotype, we addressed the question of the implication of MAPK and NF-κB pathways in the regulation
209
of CXCL10 after aggregates stimulation by using well-described pharmacological inhibitors of these
210
pathways. Our results showed that cxcl10 mRNA increased expression in response to hGH aggregates
211
was dependent on p38 MAPK, ERK, JNK and NF-κB pathways. This is consistent with findings that
212
previously reported that regulation of the cxcl10 gene was mainly controlled by NF-κB, AP-1 and ISRE
213
(interferon-stimulated response element) factors
214
cxcl10 expression have been studied in different cell lines and in response to various stimuli acting as
215
danger signals. Shen et al, showed that p38 MAPK, JNK and NF-κB played an essential role in LPS/IFN-
216
γ induction of cxcl10 expression in a mouse microglial cell line, while ERK inhibition had little effect on the
217
chemokine’s expression
218
demonstrated in human microvascular endothelial cells where CXCL10 secretion in response to TNFα
219
and IFN-γ was suppressed after pretreatment with an ERK inhibitor
220
We then showed that Rac1, member of the Rho family of small G proteins, regulated the secretion of
221
CXCL10 via ERK, JNK and p38 MAPK, while PI3K regulation of CXCL10 secretion was independent of
222
ERK, JNK and p38 MAPK pathways. Cdc42, another small G protein, did not regulate CXCL10
223
production but was involved in the MAPKs activation. The regulation of MAPKs activation by Rac1 has
224
been well documented in several cell lines, such as endothelial cells
225
murine fibroblasts
226
of CXCL10 production by Rac1. On one hand, Wang et al. showed that knockdown of Neuropilin-1, a co-
, however further investigations are needed to define
6
5
25
21
7,10
. Signaling pathways involved in the regulation of
. Nevertheless, the involvement of ERK in CXCL10 secretion has been
and airway epithelial cells
22
.
23
and human nuclear killer cells
24
,
26
. However only two other reports studied the regulation
11
227
receptor of several structurally diverse ligands, suppressed CXCL10 expression in human brain
228
microvascular endothelial cells in a Rac1-STAT1-dependent manner
229
al. showed that loss of Rac1 in keratinocytes increased the expression of CXCL10
230
of PI3K, our results were consistent with a previous study reporting that CXCL10 expression was
231
defective in the absence of PI3K regulatory subunits in embryonic fibroblasts from p85 knockout mice
232
Another report also showed that CXCL10 production in Mycobacterium bovis BCG-infected epithelial cells
233
was PI3K-dependent
234
Taken together our findings bring new insights on how protein aggregates could induce dendritic cells
235
activation. Indeed, protein aggregates can act as danger signals recognized by DC leading to the activation
236
of specific DC intracellular mechanisms involved in actin remodelling (Rac1 and PI3K) as well as in DC
237
maturation (see summary in Fig. 4).
27
. On the other hand, Pedersen et 28
. Concerning the role
29
.
30
.
12
238
ACKNOWLEDGEMENTS: Authors would like to thank Claudine Deloménie from the IPSIT
239
Transcriptomic and Proteomic Platform (TRANSPROT) for technical assistance in quantitative real-time
240
PCR.
241
FUNDING: This research did not receive any specific grant from funding agencies in the public,
242
commercial, or not-for-profit sectors.
243
CONFLICT OF INTEREST: The authors state no conflict of interest.
244
SUPPLEMENTARY DATA: This article contains supplementary material available from the authors by
245
request.
13
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Mainiero F, Soriani A, Strippoli R, et al. RAC1/P38 MAPK signaling pathway controls beta1 integrin-
Rul W, Zugasti O, Roux P, et al. Activation of ERK, controlled by Rac1 and Cdc42 via Akt, is required for anoikis. Ann N Y Acad Sci. 2002;973:145-148.
26.
Terakado M, Gon Y, Sekiyama A, et al. The Rac1/JNK pathway is critical for EGFR-dependent
306
barrier formation in human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol.
307
2011;300(1):L56-63.
308
27.
309 310
brain microvascular endothelial cells. J Cell Sci. 2016;129(20):3911-3921. 28.
311 312
Pedersen E, Wang Z, Stanley A, et al. RAC1 in keratinocytes regulates crosstalk to immune cells by Arp2/3-dependent control of STAT1. J Cell Sci. 2012;125(22):5379-5390. doi:10.1242/jcs.107011
29.
313 314
Wang Y, Cao Y, Mangalam AK, et al. Neuropilin-1 modulates interferon-γ-stimulated signaling in
Kaur S, Sassano A, Joseph AM, et al. Dual Regulatory Roles of the Phosphatidylinositol 3-Kinase In Interferon Signaling. J Immunol. 2008;181(10):7316-7323.
30.
Méndez-Samperio P, Pérez A, Rivera L. Mycobacterium bovis Bacillus Calmette-Guérin (BCG)-
315
induced activation of PI3K/Akt and NF-kB signaling pathways regulates expression of CXCL10 in
316
epithelial cells. Cell Immunol. 2009;256(1-2):12-18.
317
16
318
FIGURE LEGENDS
319
Figure 1. hGH aggregates induce p38 MAPK, ERK and JNK phosphorylation in stimulated moDC and
320
trigger NF-κB p65 subunit nuclear translocation.
321
Immature moDC were stimulated for 30 min with native (N) or aggregated (A) hGH (50 µg mL ). (a)
322
Immunoblotting of whole-cell extracts was used to quantify the phosphorylated forms of p38 MAPK, ERK,
323
JNK and NF-κB p65 subunit. The results of 5 independent experiments are shown, with values for each
324
donor represented by one given symbol. (b) p65 DNA-binding activity was evaluated using an
325
oligonucleotide pull-down assay. The results of 4 independent experiments are shown, with values for
326
each donor represented by one given symbol. Bands were quantified using ImageLab software. Results
327
are expressed as the fold induction, representing the ratio of the normalized intensity of specific bands of
328
treated cells divided by the normalized intensity of specific bands of untreated cells. *: P < 0.05; **: P <
329
0.01, Mann-Whitney U-test. NS: non-stimulated cells.
-1
330 331
Figure 2. Regulation of p38 MAPK, ERK and JNK activation by RhoGTPases & PI3K in moDC treated
332
with hGH aggregates.
333
Immature moDC were pretreated for 1 h with either Ly294002 (25 µM), ML141 (10 µM) or Rac1 inhibitor
334
(50 µM) and then stimulated for 30 min with native (N) hGH or aggregates (A) (50 µg mL ).
335
Immunoblotting of whole-cell extracts was used to quantify the phosphorylated forms of p38 MAPK, ERK
336
and JNK. (a) Representative Western blotting results, showing bands for phosphorylated p38 MAPK (P-
337
p38), ERK1/2 (P-ERK), JNK (P-JNK) and total p38 MAPK. (b) Bands were quantified using ImageLab
338
software. Results are expressed as fold induction. For cells treated with N GH or A GH, folds are
339
calculated as the ratio of the normalized intensity of specific bands of treated cells divided by the
340
normalized intensity of specific bands of untreated cells. For cells treated with inhibitors and A GH, folds
341
are calculated as the ratio of the normalized intensity of specific bands of treated cells divided by the
342
normalized intensity of specific bands of cells treated with the corresponding inhibitor alone. The results
343
of 4 independent experiments are shown, with values for each donor represented by one given symbol. *:
344
P < 0.05, Mann-Whitney U-test. NS: non-stimulated cells.
345
Figure 3. Implication of hGH aggregate-induced signaling pathways in CXCL10 production by moDC.
-1
17
346
(a) MAPK and NF-κB pathways implication in aggregate-mediated cxcl10 mRNA expression. Immature
347
moDC were pretreated for 1 h with Bay 11-7085 (3 µM) or for 30 min with either SP600125 (20 µM),
348
PD98059 (20 µM) or SB203580 (20 µM) and then stimulated for 4 h with native (N) or aggregated (A)
349
hGH (50 µg mL ). Cxcl10 mRNA levels were quantified by real-time RT-PCR. Results were normalized to
350
non-stimulated moDC and corrected by the expression of β-actin and gapdh. The results of 4
351
independent experiments are shown, with values for each donor represented by one given symbol. *: P <
352
0.05, Mann-Whitney U-test. NS: non-stimulated cells.
353
(b) Rho GTPases and PI3K implication in CXCL10 secretion induced by hGH aggregates. Immature
354
moDCs were pretreated for 1 h with either Ly294002 (25 µM), ML141 (10 µM) or Rac1 inhibitor (50 µM)
355
and then stimulated for 24 h with hGH aggregates (A) (50 µg mL ). CXCL10 concentration in culture
356
supernatants was analyzed in duplicate using an electroluminescence assay. The results of 4
357
independent experiments are shown, with values for each donor represented by one given symbol. *: P <
358
0.05, Mann-Whitney U-test.
-1
-1
359 360
Figure 4. Growth hormone aggregates induced CXCL10 production in human dendritic cells via Rac1
361
and PI3K signaling pathways.
362
We previously showed that growth hormone aggregates induced dendritic cells maturation with a notably
363
strong production of CXCL10, a T-lymphocyte chemoattractant. Indeed this maturation was sufficient to
364
drive an allogeneic CD4+ T-cell response towards a Th1 polarization . Here we showed that the
365
increased secretion of CXCL10 in response to hGH aggregates is regulated by Rac1 pathway via p38
366
MAPK, JNK and ERK and by NF-κB and PI3K pathways.
5
367 368
Supplementary figure S1: Immature moDC were pretreated for 1 h with either Ly294002 (25 µM),
369
ML141 (10 µM) or Rac1 inhibitor (50 µM) and then stimulated or not for 30 min with native (N) hGH or
370
aggregates (A) (50 µg mL ). Immunoblotting of whole-cell extracts was used to quantify the
371
phosphorylated forms of p38 MAPK, ERK and JNK. Bands were quantified using ImageLab software.
372
Results are expressed as the fold induction, representing the ratio of the normalized intensity of specific
-1
18
373
bands of treated cells divided by the normalized intensity of specific bands of untreated cells. The results
374
of 4 independent experiments are shown.
19
S0022-3549(19)30577-5
DOI:
https://doi.org/10.1016/j.xphs.2019.09.007
Reference:
XPHS 1708
To appear in:
Journal of Pharmaceutical Sciences
Received Date: 14 June 2019 Revised Date:
30 August 2019
Accepted Date: 6 September 2019
Please cite this article as: Nabhan M, Gallais Y, Pallardy M, Turbica I, Growth hormone aggregates activation of human dendritic cells is controlled by Rac1 and PI3 kinase signaling pathways, Journal of Pharmaceutical Sciences (2019), doi: https://doi.org/10.1016/j.xphs.2019.09.007. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
1
Growth hormone aggregates activation of human dendritic cells is controlled by Rac1 and PI3
2
kinase signaling pathways
3
Myriam Nabhan , Yann Gallais , Marc Pallardy and Isabelle Turbica
4
1 Inflammation Chimiokines et Immunopathologie, INSERM, Fac. de pharmacie - Univ.Paris-Sud,
5
Université Paris-Saclay, Châtenay-Malabry, France.
6
2 Formerly: Inflammation Chimiokines et Immunopathologie, INSERM, Fac. de pharmacie - Univ.Paris-
7
Sud, Université Paris-Saclay, Châtenay-Malabry, France.
8
Current address: CEA-Saclay, Institute Frederic Joliot, Service d'Ingénierie Moléculaire des Protéines,
9
Gif-sur-Yvette, France
1,§
2,§
1
1*
10 11
§: Both authors contributed equally to this work.
12 13
* Corresponding Author: [email protected]; +33(0)146835299
14
KEYWORDS: protein aggregation, immunogenicity, immune response, in vitro model(s), cell biology
15
SHORT TITLE: CXCL10 regulation by growth hormone aggregates
1
16
ABSTRACT
17
The presence of protein aggregates in biological products is suggested to promote immunogenicity,
18
leading to the production of anti-drug antibodies with neutralizing capacities. This suggests a CD4 T-cell
19
dependent adaptive immune response thus a pivotal role for antigen presenting cells, such as dendritic
20
cells (DC). We previously showed that human growth hormone (hGH) aggregates induced DC maturation,
21
with notably an increase in CXCL10 production. DC phenotypic modifications were sufficient to promote
22
allogeneic CD4+ T-cell proliferation with Th1 polarization. In this work, we identified the main intracellular
23
signaling pathways involved in DC activation by hGH aggregates, showing that aggregates induced p38
24
MAPK, ERK and JNK phosphorylation, as well as NF-κB subunit p65 nuclear translocation. Next,
25
investigating the implication of Rho GTPases and phosphoinositide 3-kinase (PI3K) in activated DC
26
showed that Rac1 and Cdc42 regulated the phosphorylation of MAP kinases, whereas PI3K was only
27
implicated in JNK phosphorylation. Furthermore, we showed that Rac1 and PI3K pathways, but not
28
Cdc42, regulated the production of CXCL10 via the MAP kinases and NF-κB. Taken together our results
29
bring new insight on how protein aggregates could induce DC activation, leading to a better understanding
30
of aggregates role in therapeutic proteins immunogenicity.
+
31 32
Abbreviations: DC: dendritic cells; ERK: extracellular signal-regulated kinase; hGH: human growth
33
hormone; JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; moDC: monocyte-
34
derived dendritic cells; NF-κB: nuclear factor κB ; PI3K: phosphoinositide-3 kinase; Th: T helper.
2
35
INTRODUCTION
36
Patients treated with biological products (BP) frequently develop anti-drug antibodies (ADA) with
37
potentially neutralizing capacities leading to loss of clinical response
38
BP have been suggested as one of the factors promoting immunogenicity, as their occurrence seems to
39
be correlated with ADA development in mice models
40
dependent adaptive immune response and therefore a pivotal role for antigen presenting cells, such as
41
dendritic cells (DC) 3. We, and others, have previously shown that protein aggregates could act as a danger
42
signal and be recognized by dendritic cells inducing their maturation and CD4 T-cell response
43
previous work, we demonstrated that hGH aggregates were able to induce monocyte-derived DC (moDC)
44
maturation, showed by a significant increase of surface markers expression and a high production of pro-
45
inflammatory cytokines and inflammatory chemokines with notably an increase in CXCL10 secretion
46
compared to the native protein. Moreover, moDC phenotype induced by the hGH aggregates was able to
47
drive allogeneic CD4 T cells towards a T helper 1 (Th1) profile. In this previous work, aggregates were
48
generated by submitting native human growth hormone (hGH, Somatropin) to a stir stress .
49
CXCL10, also called IP-10 (interferon γ-induced protein 10), is an inflammatory chemokine that binds to
50
CXCR3, a receptor expressed on a wide range of immune cells including T lymphocytes, natural killer
51
cells (NK), DC and macrophages, thus favoring their activation and recruitment
52
upregulates T-bet expression as well as IFN-γ (interferon-γ) production in CD4 T cells thus promoting
53
Th1 polarization . The secretion of CXCL10 in response to external stimuli is dependent on an array of
54
transcription factors including NF-κB (nuclear factor κB), AP-1 (activator protein 1) and ISRE (interferon-
55
stimulated response element)
56
pathways that could be involved in particles internalization and actin cytoskeleton reorganization
57
Following these observations, we raised the hypothesis that protein aggregates could trigger specific
58
signaling pathways in human dendritic cells. hGH aggregates could also act differently than aggregates
59
from therapeutic antibodies usually used as a model. In the present work, we searched for pathways that
60
could be rapidly activated by these aggregates. CXCL10 is an important chemokine for T-cell biology and
61
in our hand it is produced by moDC treated with hGH aggregates but not with therapeutic antibody
62
aggregates
1,2
. The presence of aggregates in
3,4
+
. Production of ADA suggests a CD4 T-cell
+
5,6
. In a
+
5
7,8
. Moreover, CXCL10
+
9
10
. These transcription factors are regulated by upstream signaling 11
.
12
. CXCL10 can then be used as a specific biomarker of DC activation by GH-type
3
63
aggregates. For the first time, our results highlighted the involvement of small GTPases and of
64
phosphoinositide-3 kinase (PI3K) in the regulation of the signaling pathways induced by aggregates in
65
moDC.
4
66
MATERIAL AND METHODS
67
Samples preparation
68
Lyophilized human Growth Hormone (hGH) was purchased from European Pharmacopoeia (ref:
69
S0947000; EDQM Council of Europe, Strasbourg, France) and diluted to a final concentration of 1 mg mL
70
1
71
samples were stirred at 1000 rpm for 72 hours at room temperature in 5 mL glass tubes with an 8x2 mm
72
Teflon stirrer bar. Endotoxin was quantified for native and aggregated hGH by using a colorimetric assay
73
(ToxinSensor, Genscript, Paris, France), according to the manufacturer’s instructions.
74
Generation of human monocyte-derived dendritic cells (moDC)
75
Peripheral blood mononuclear cells (PBMC) were purified from buffy coats obtained from Etablissement
76
Français du Sang (EFS Rennes, France) by density centrifugation on a Ficoll gradient (lymphocyte
77
separation medium; GE Healthcare, Buc, France). Healthy donors gave their written consent for the use
78
of blood donation for research purposes. MoDC preparation was previously described .
79
Treatment of moDC
80
After differentiation, moDC were stimulated with 50 µg mL
81
times. In some experiments, moDC were pretreated for 30 min with PD98059 (20 µM), SP600125 (20
82
µM) or SB203580 (20 µM) and for 1 h with Bay 11-7085 (3 µM). For Rho GTPases and PI3K
83
experiments, moDC were pretreated for 1h with Ly294002 (25 µM), Rac1 inhibitor (50 µM) or ML141 (10
84
µM). All inhibitors (diluted in DMSO) were purchased from Calbiochem, Darmstadt, Germany.
85
Quantitative real-time RT-PCR assay of CXCL10 in moDC
86
Total RNA was extracted after lysis of treated cells pretreated or not with inhibitors, using the Nucleospin
87
RNA kit (Macherey Nagel, Hoerdt, France), according to the manufacturer’s instructions. First-strand
88
cDNA was synthesized from total RNA, as previously described
89
performed by use of the SYBR Green technology on a CFX96 system (Bio-Rad Laboratories, Marnes la
90
Coquette, France), as previously described
91
CFX Manager software. Ratios were calculated as the geometrical mean of (1 + E)
92
the efficiency and ∆∆Ct is the target gene expression of treated cells compared with untreated cells,
93
corrected to the expression of the reference genes gapdh and β-actin. Results were expressed as the
-
5
with sterile PBS. Aggregates preparation by stir stress was previously described . In brief, 1 mL
5
-1
of native or aggregated hGH for indicated
13
. Real-time quantitative PCR was
13
. Quantification was performed with Bio-Rad Laboratories -∆∆Ct
values, where E is
5
-∆∆Ct
-∆∆Ct
94
fold-factor induction [ratio of (1 + E)
95
specific primers, purchased from Eurofins Genomics (Ebersberg, Germany), were used (forward and
96
reverse, respectively): cxcl10: 5’-TCT AAG TGG CAT TCA AGG AGT ACC-3’ and 5’-AAA GAC CTT GGA
97
TTA ACA GGT TGA-3’ ; gapdh: 5’-CAG CCT CAA GAT CAT CAG CA-3’ and 5’-TGT CGT CAT GAG
98
TCC TTC CA-3’; β-actin: 5’-GGC ATC CTC ACC CTG AAG TA-3’ and 5’-GCA CAC GCA GCT CAT TG
99
TAG-3’.
of treated cells/(1 + E)
of untreated cells]. The following
100
Immunoblot analysis of signaling pathways in moDC
101
After pretreatment or not with inhibitors, moDC were incubated for 30 min with native or aggregated hGH,
102
harvested and washed in cold phosphate buffered saline before lysis. 30 micrograms of denatured protein
103
were loaded onto 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes, which
104
were successively incubated with antibodies directed against the phosphorylated forms of JNK
105
(Thr183/Tyr185), p38 MAPK (Thr180/Tyr182), ERK1/2 (Thr202/Tyr204) and the NF-κB p65 subunit
106
(Ser536) and an antibody against total p38 MAPK as a loading control (Ozyme, Montigny-Le-Bretonneux,
107
France). Immunoreactive bands were detected by their chemiluminescence by use of the ChemiDoc
108
XRS+ System (Bio-Rad Laboratories, Marnes-La-Coquette, France). Bands were quantified with
109
ImageLab software. In some experiments, NF-κB activation was also evaluated by a pull-down assay, as
110
described previously
111
lysed in NP-40 lysis hypertonic buffer. Whole-cell extracts (150 µg) were incubated at 4°C for 90 min with
112
2 µg double-stranded 59-biotinylated oligonucleotide coupled to 30 mL streptavidin-agarose beads
113
(Sigma-Aldrich, St-Quentin Fallavier, France). Complexes were washed in binding buffer and eluted by
114
boiling in reducing sample buffer before protein separation by SDS-PAGE gel, followed by Western blot
115
analysis of the NF-κB p65 subunit, as described above.
116
CXCL10 quantification in moDC supernatants
117
After pretreatment or not with inhibitors, moDC were stimulated for 24 h with native or aggregated hGH.
118
Meso Scale Discovery (MSD, Rockville, Maryland, USA) U-plex assay was performed on culture
119
supernatants according to the manufacturer’s instructions to measure CXCL10 (IP-10). The quantification
120
range for CXCL10 is: 2.2–9040 pg mL .
121
Statistical analysis
14
. In brief, after a 30 min incubation with native or aggregated hGH, moDC were
-1
6
122
Data are expressed as means ± SEM. Differences between groups were evaluated with the Mann–
123
Whitney U-test (Prism software, GraphPad, La Jolla, CA). P-values below 0.05 were considered to denote
124
statistical significance.
7
125
RESULTS
126
MAPKs and NF-κB are activated in response to hGH aggregates
127
In order to study the involvement of mitogen-activated protein kinase (MAPKs) and NF-κB pathways in
128
moDC in response to aggregates, we first analyzed their phosphorylation by Western blotting. Native
129
hGH had little effect on proteins phosphorylation after 30 min of incubation compared to non-stimulated
130
cells (Fig. 1(a)). However, aggregated hGH significantly increased phosphorylation of p38 MAPK,
131
extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and NF-κB subunit p65 (Fig.
132
1(a)). Using a pull-down assay, we showed an increased NF-κB p65 DNA-binding activity after 30 min of
133
treatment with aggregated hGH compared to native hGH (Fig. 1(b)). The overall results confirmed the
134
involvement of p38 MAPK, ERK, JNK and NF- κB in the intracellular signaling mediated by hGH
135
aggregates in moDC.
136
Control of p38 MAPK, ERK & JNK activation by Rho GTPases & PI3K
137
Particles and aggregates are known to provoke cell membrane alterations leading to cytoskeleton
138
reorganization and small G proteins activation
139
MAPKs activation following hGH aggregates treatment by using pharmacological inhibitors of these small
140
G proteins (Rac1 inhibitor and ML141, a Cdc42 inhibitor). Cells were pretreated for 1 h with these
141
inhibitors and then stimulated for 30 min with hGH aggregates. We first assessed the effect of the
142
inhibitors alone. Indeed, Cdc42 inhibitor alone slightly increased the phosphorylation of ERK whereas
143
PI3K inhibitor alone slightly increased JNK phosphorylation (Supplementary Fig. S1). Then, we
144
determined the effect of hGH aggregates on MAPKs activation, by taking into account the by stander
145
effect of the inhibitors (Fig.2). We observed that p38 MAPK and ERK phosphorylation was significantly
146
decreased by the inhibitors of Cdc42 and Rac1 whereas JNK phosphorylation showed a trend to
147
decrease with Cdc42 inhibitor (Fig. 2 (a) and (b)).
148
Knowing that PI3K is also involved in controlling cytoskeletal changes upon large particles’
149
internalization , we investigated PI3K impact on the MAPKs activation in response to hGH aggregates.
150
Cells were pretreated for 1 h with a PI3K inhibitor (Ly294002) and then stimulated for 30 min with hGH
151
aggregates. We observed that p38 MAPK and ERK phosphorylation was not altered by the PI3K inhibitor.
15
. Therefore, we assessed if these proteins could regulate
15
8
152
However, JNK phosphorylation showed a trend to decrease after pretreatment with this inhibitor (Fig. 2
153
(a) and (b)).
154
hGH aggregates regulate cxcl10 mRNA expression via MAPK and NF-κB pathways
155
In order to connect the upregulation of MAPKs and NF-κB signaling pathways induced by aggregated
156
with cellular responses, we used well-described pharmacological inhibitors of these pathways and
157
investigated their implication in cxcl10 expression. At day 4, cells were pretreated for 1 h with Bay 117085
158
(a NF-kB inhibitor), or for 30 min with either PD98059 (an ERK inhibitor), SP600125 (a JNK inhibitor) and
159
SB203580 (a p38 MAPK inhibitor) and then stimulated for 4 h with hGH aggregates. Inhibitors
160
concentrations and treatment timelines were optimized to avoid cellular toxicity, according to previous
161
studies
162
inhibitors, suggesting that hGH aggregates modulate cxcl10 expression via NF-κB, ERK, JNK and p38
163
MAPK signaling pathways (Fig. 3(a)).
164
Rac1 and PI3K control hGH aggregates-mediated production of CXCL10
165
Next, using the pharmacological inhibitors of Rho GTPases and PI3K, we analyzed the impact of Rac1,
166
Cdc42 and PI3K on CXCL10 secretion in aggregates-treated moDC. Cells were pretreated for 1 h with
167
inhibitors and then stimulated for 24 hours with hGH aggregates. We observed that PI3K and Rac1
168
inhibitors downregulated CXCL10 production, whereas Cdc42 inhibitor had little effect on CXCL10
169
secretion (Fig. 3(b)). These results suggest that PI3K and Rac1 are involved in DC activation induced by
170
hGH aggregates
16
. Results showed that cxcl10 expression was decreased when moDC were pretreated with these
9
171
DISCUSSION
172
Aggregation of therapeutic proteins has been associated with an increased susceptibility to develop an
173
immune response targeting the BP
174
immune danger signal and initiate a protein-specific adaptive immune response, we analyzed the impact
175
of hGH aggregates on the regulation of the intracellular mechanisms leading to moDC maturation. We
176
had previously shown that hGH aggregates, generated under stir stress, induced moDC maturation,
177
increasing the expression of the surface markers CD80, CD83, CD86 and HLA-DR as well as the
178
secretion of the cytokines IL-6, IL-8, IL-12p40, and the chemokines CCL2, CCL3, CCL4 and CXCL10 . In
179
the present study, we confirmed this inflammatory phenotype and used the production of the inflammatory
180
chemokine CXCL10 as a biomarker of moDC activation in response to hGH aggregates. The choice to
181
focus on CXCL10 was made for two main reasons: first, it is an important chemokine playing a central
182
role in T-helper cell polarization and second, we described its production in response to hGH aggregates
183
but not with therapeutic antibody aggregates
184
To study intracellular mechanisms regulated in aggregates-stimulated moDC, we first showed that
185
aggregates increased phosphorylation of p38 MAPK, ERK, JNK as well as NF-κB subunit p65 binding.
186
This observation seems in agreement with a recent study by Polumuri et al. also showing the implication
187
of intravenous immunoglobulin (IVIG) and bevacizumab aggregates (shaken or stirred) in the activation of
188
MAPKs in human peripheral blood mononuclear cells
189
phosphorylation, but not p38 or JNK, was induced in moDC in response to infliximab aggregates
190
Previous reports also demonstrated activation of MAPKs and NF-κB subunit p65 in moDC in response to
191
danger signals such as lipopolysaccharide (LPS) and contact sensitizers, such as nickel
192
then interested in the upstream signaling pathways that participated in DC response to hGH aggregates.
193
Both Rac1 and PI3K are highly involved in actin remodeling observed in phagocytosis, the internalization
194
mechanism of large insoluble particles by innate immune cells such as macrophages, neutrophils and
195
dendritic cells
196
to hGH aggregates and these aggregates, having a diameter larger than 0.5 µm , induced cytoskeletal
197
changes leading to their possible internalization via phagocytosis. A previous report on sub-visible
198
aggregates effect on moDC showed that stir-stressed rituximab was efficiently uptaken by moDC and
3,17
. In order to further understand how protein aggregates act as an
5
12
.
18
. Furthermore, we recently showed that ERK 12
.
14,16,19
. We were
11
. Our observations suggest that Rac1 and PI3K are implicated in the response of moDC 5
10
20
199
rapidly accumulated in late endosomes
200
aggregates recognition and internalization mechanisms by DC. The involvement of small G protein
201
signalization suggests that the cell membrane could somehow be affected by GH particulates. Therefore,
202
it is possible that GH aggregates could follow different pathways than those described for antibodies
203
aggregates with involvement of Toll-like receptors and Fc receptors . However, it is unlikely that GH
204
aggregates bind to GH receptors expressed on moDC since aggregates formation often requires
205
conformational misfolding of the monomers to favor interactions. Indeed, we previously showed that GH
206
aggregates formation induced a loss of α-helix in secondary structure .
207
In order to link the intracellular events upregulated by hGH aggregates with the inflammatory moDC
208
phenotype, we addressed the question of the implication of MAPK and NF-κB pathways in the regulation
209
of CXCL10 after aggregates stimulation by using well-described pharmacological inhibitors of these
210
pathways. Our results showed that cxcl10 mRNA increased expression in response to hGH aggregates
211
was dependent on p38 MAPK, ERK, JNK and NF-κB pathways. This is consistent with findings that
212
previously reported that regulation of the cxcl10 gene was mainly controlled by NF-κB, AP-1 and ISRE
213
(interferon-stimulated response element) factors
214
cxcl10 expression have been studied in different cell lines and in response to various stimuli acting as
215
danger signals. Shen et al, showed that p38 MAPK, JNK and NF-κB played an essential role in LPS/IFN-
216
γ induction of cxcl10 expression in a mouse microglial cell line, while ERK inhibition had little effect on the
217
chemokine’s expression
218
demonstrated in human microvascular endothelial cells where CXCL10 secretion in response to TNFα
219
and IFN-γ was suppressed after pretreatment with an ERK inhibitor
220
We then showed that Rac1, member of the Rho family of small G proteins, regulated the secretion of
221
CXCL10 via ERK, JNK and p38 MAPK, while PI3K regulation of CXCL10 secretion was independent of
222
ERK, JNK and p38 MAPK pathways. Cdc42, another small G protein, did not regulate CXCL10
223
production but was involved in the MAPKs activation. The regulation of MAPKs activation by Rac1 has
224
been well documented in several cell lines, such as endothelial cells
225
murine fibroblasts
226
of CXCL10 production by Rac1. On one hand, Wang et al. showed that knockdown of Neuropilin-1, a co-
, however further investigations are needed to define
6
5
25
21
7,10
. Signaling pathways involved in the regulation of
. Nevertheless, the involvement of ERK in CXCL10 secretion has been
and airway epithelial cells
22
.
23
and human nuclear killer cells
24
,
26
. However only two other reports studied the regulation
11
227
receptor of several structurally diverse ligands, suppressed CXCL10 expression in human brain
228
microvascular endothelial cells in a Rac1-STAT1-dependent manner
229
al. showed that loss of Rac1 in keratinocytes increased the expression of CXCL10
230
of PI3K, our results were consistent with a previous study reporting that CXCL10 expression was
231
defective in the absence of PI3K regulatory subunits in embryonic fibroblasts from p85 knockout mice
232
Another report also showed that CXCL10 production in Mycobacterium bovis BCG-infected epithelial cells
233
was PI3K-dependent
234
Taken together our findings bring new insights on how protein aggregates could induce dendritic cells
235
activation. Indeed, protein aggregates can act as danger signals recognized by DC leading to the activation
236
of specific DC intracellular mechanisms involved in actin remodelling (Rac1 and PI3K) as well as in DC
237
maturation (see summary in Fig. 4).
27
. On the other hand, Pedersen et 28
. Concerning the role
29
.
30
.
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ACKNOWLEDGEMENTS: Authors would like to thank Claudine Deloménie from the IPSIT
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Transcriptomic and Proteomic Platform (TRANSPROT) for technical assistance in quantitative real-time
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PCR.
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FUNDING: This research did not receive any specific grant from funding agencies in the public,
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commercial, or not-for-profit sectors.
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CONFLICT OF INTEREST: The authors state no conflict of interest.
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SUPPLEMENTARY DATA: This article contains supplementary material available from the authors by
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request.
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FIGURE LEGENDS
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Figure 1. hGH aggregates induce p38 MAPK, ERK and JNK phosphorylation in stimulated moDC and
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trigger NF-κB p65 subunit nuclear translocation.
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Immature moDC were stimulated for 30 min with native (N) or aggregated (A) hGH (50 µg mL ). (a)
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Immunoblotting of whole-cell extracts was used to quantify the phosphorylated forms of p38 MAPK, ERK,
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JNK and NF-κB p65 subunit. The results of 5 independent experiments are shown, with values for each
324
donor represented by one given symbol. (b) p65 DNA-binding activity was evaluated using an
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oligonucleotide pull-down assay. The results of 4 independent experiments are shown, with values for
326
each donor represented by one given symbol. Bands were quantified using ImageLab software. Results
327
are expressed as the fold induction, representing the ratio of the normalized intensity of specific bands of
328
treated cells divided by the normalized intensity of specific bands of untreated cells. *: P < 0.05; **: P <
329
0.01, Mann-Whitney U-test. NS: non-stimulated cells.
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330 331
Figure 2. Regulation of p38 MAPK, ERK and JNK activation by RhoGTPases & PI3K in moDC treated
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with hGH aggregates.
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Immature moDC were pretreated for 1 h with either Ly294002 (25 µM), ML141 (10 µM) or Rac1 inhibitor
334
(50 µM) and then stimulated for 30 min with native (N) hGH or aggregates (A) (50 µg mL ).
335
Immunoblotting of whole-cell extracts was used to quantify the phosphorylated forms of p38 MAPK, ERK
336
and JNK. (a) Representative Western blotting results, showing bands for phosphorylated p38 MAPK (P-
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p38), ERK1/2 (P-ERK), JNK (P-JNK) and total p38 MAPK. (b) Bands were quantified using ImageLab
338
software. Results are expressed as fold induction. For cells treated with N GH or A GH, folds are
339
calculated as the ratio of the normalized intensity of specific bands of treated cells divided by the
340
normalized intensity of specific bands of untreated cells. For cells treated with inhibitors and A GH, folds
341
are calculated as the ratio of the normalized intensity of specific bands of treated cells divided by the
342
normalized intensity of specific bands of cells treated with the corresponding inhibitor alone. The results
343
of 4 independent experiments are shown, with values for each donor represented by one given symbol. *:
344
P < 0.05, Mann-Whitney U-test. NS: non-stimulated cells.
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Figure 3. Implication of hGH aggregate-induced signaling pathways in CXCL10 production by moDC.
-1
17
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(a) MAPK and NF-κB pathways implication in aggregate-mediated cxcl10 mRNA expression. Immature
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moDC were pretreated for 1 h with Bay 11-7085 (3 µM) or for 30 min with either SP600125 (20 µM),
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PD98059 (20 µM) or SB203580 (20 µM) and then stimulated for 4 h with native (N) or aggregated (A)
349
hGH (50 µg mL ). Cxcl10 mRNA levels were quantified by real-time RT-PCR. Results were normalized to
350
non-stimulated moDC and corrected by the expression of β-actin and gapdh. The results of 4
351
independent experiments are shown, with values for each donor represented by one given symbol. *: P <
352
0.05, Mann-Whitney U-test. NS: non-stimulated cells.
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(b) Rho GTPases and PI3K implication in CXCL10 secretion induced by hGH aggregates. Immature
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moDCs were pretreated for 1 h with either Ly294002 (25 µM), ML141 (10 µM) or Rac1 inhibitor (50 µM)
355
and then stimulated for 24 h with hGH aggregates (A) (50 µg mL ). CXCL10 concentration in culture
356
supernatants was analyzed in duplicate using an electroluminescence assay. The results of 4
357
independent experiments are shown, with values for each donor represented by one given symbol. *: P <
358
0.05, Mann-Whitney U-test.
-1
-1
359 360
Figure 4. Growth hormone aggregates induced CXCL10 production in human dendritic cells via Rac1
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and PI3K signaling pathways.
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We previously showed that growth hormone aggregates induced dendritic cells maturation with a notably
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strong production of CXCL10, a T-lymphocyte chemoattractant. Indeed this maturation was sufficient to
364
drive an allogeneic CD4+ T-cell response towards a Th1 polarization . Here we showed that the
365
increased secretion of CXCL10 in response to hGH aggregates is regulated by Rac1 pathway via p38
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MAPK, JNK and ERK and by NF-κB and PI3K pathways.
5
367 368
Supplementary figure S1: Immature moDC were pretreated for 1 h with either Ly294002 (25 µM),
369
ML141 (10 µM) or Rac1 inhibitor (50 µM) and then stimulated or not for 30 min with native (N) hGH or
370
aggregates (A) (50 µg mL ). Immunoblotting of whole-cell extracts was used to quantify the
371
phosphorylated forms of p38 MAPK, ERK and JNK. Bands were quantified using ImageLab software.
372
Results are expressed as the fold induction, representing the ratio of the normalized intensity of specific
-1
18
373
bands of treated cells divided by the normalized intensity of specific bands of untreated cells. The results
374
of 4 independent experiments are shown.
19