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\begin{document}

\title{Increased expression of CXCL2 in ACPA-positive rheumatoid arthritis and
its role in osteoclastogenesis}



\author[1]{Lin Sun}%
\author[1]{Xinyu Wang}%
\author[2]{Ning He}%
\author[1]{Zhuo An}%
\author[1]{Ruohan Yu}%
\author[1]{Changhong Li}%
\author[2]{Yanming Li}%
\author[2]{Yongjun Li}%
\author[1]{Xiangyuan Liu}%
\author[2]{dong Fang}%
\author[1]{Jinxia Zhao}%
\affil[1]{Peking University Third Hospital}%
\affil[2]{Beijing Institute of Genomics Chinese Academy of Sciences}%


\vspace{-1em}



  
  \date{May 16, 2024}


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\selectlanguage{english}
\begin{abstract}
Background. Anti-citrullinated protein/peptide antibodies (ACPA) play
important roles in the pathogenesis of rheumatoid arthritis (RA).
ACPA-positive (ACPA+) and ACPA-negative (ACPA-) RA were suggested to be
different disease subsets with distinct differences in genetic variation
and clinical outcomes. The aims of the present study were to compare
gene expression profiles in ACPA+ and ACPA- RA and identify novel
candidate gene signatures that might serve as therapeutic targets.
Methods. Comprehensive transcriptome analysis of peripheral blood
mononuclear cells (PBMCs) from ACPA+ and ACPA- RA patients, and healthy
controls was performed via RNA sequencing. A validation cohort was used
to further investigate differentially expressed genes via PCR and ELISA.
Spearman's correlation test was used to evaluate the correlation of
differentially expressed genes and the clinical and laboratory data of
the patients. The role of differentially expressed genes in
osteoclastogenesis was further investigated. Results. Expression of
C-X-C motif chemokine ligand 2 (CXCL2) was significantly increased in
ACPA+ RA than in ACPA- RA, which was validated in PBMCs and serum. CXCL2
promoted the migration of CD14+ monocytes and increased
osteoclastogenesis in RA patients. RAW264.7 macrophages were used to
investigate specific mechanisms, and the results suggested that CXCL2
stimulated osteoclastogenesis via ERK MAPK and NF\selectlanguage{greek}κ\selectlanguage{english}B pathways.
Conclusion. CXCL2 was highly expressed in ACPA+ RA than in ACPA- RA.
CXCL2 promoted osteoclastogenesis and was related to bone erosion in RA,
which suggest that the blockade of CXCL2 might be a novel strategy for
the treatment of RA.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Rheumatoid arthritis (RA) is a systemic autoimmune disease that involves
activation of inflammatory cells, synovial hyperplasia, and destruction
of cartilage and bone(1). In recent years, anti-citrullinated
protein/peptide antibodies (ACPAs) have been used as biomarkers in the
diagnosis of RA, and to predict clinical outcomes(2-4). Based on the
presence or absence of ACPA, RA can be classified into two subtypes as
ACPA-positive (ACPA+) or ACPA-negative (ACPA-). It has been reported
that ACPAs are detectable years before the onset of arthritis, and they
are strong predictors of progression to classic RA in patients with
undifferentiated arthritis(5, 6). Moreover, ACPAs are associated with RA
severity. RA patients with ACPAs exhibit more severe radiographic damage
than those without ACPAs(7-10). Many studies have suggested that ACPAs
play a pathophysiologic role in RA(11). In recent years, a number of
candidate genes have been shown to be differentially associated with
susceptibility in ACPA+ and ACPA-RA(12-14). With the advent of
next-generation sequencing technologies such as RNA sequencing, more
comprehensive and accurate transcriptome analysis has become feasible
and affordable. In RNA sequencing, short fragments of complementary DNA
(cDNA) are sequenced (`reads') then mapped onto the reference genome.
RNA sequencing facilitates both the identification of differentially
expressed genes and precise quantitative determination of exon and
isoform (alternative splicing) expression, along with the
characterisation of transcription initiation sites and new splicing
variants(15, 16).

In the present study, comprehensive transcriptome analysis of PBMCs from
ACPA+ and ACPA- RA patients was performed to identify novel candidate
gene signatures that might be involved in the pathogenesis of different
subsets of disease.

\textbf{Materials and Methods}

\textbf{Patients}

Two ACPA+ and two ACPA- RA patients from Peking University Third
Hospital who fulfilled the 2010 European League Against
Rheumatism/American College of Rheumatology classification criteria for
RA(2) were enrolled in the study. All 4 patients were newly diagnosed,
treatment naive, menopausal, and non-smoker women from the northern Han
population with symptom duration of less than 6 months, and none had any
other chronic diseases. Three matched healthy individuals were included
in the study.

PBMC collected from 40 ACPA+, 40 ACPA- RA patients, and 40 healthy
controls were applied for quantitative PCR to validate candidate genes
expression. Serum CXCL2 levels were detected by ELISA in a cohort
consisted of 70 ACPA+, 37 ACPA- RA patients, and 40 healthy controls.
The clinical and laboratory data of the patients were collected from
electronic medical records. In all comparisons mentioned, the groups
were age and sex matched. The present study was conducted in accordance
with the Declaration of Helsinki. The Ethics Committee of Peking
University Third Hospital approved the study. All procedures involving
specimens obtained from humans were performed with informed consent from
each patient.

\textbf{RNA extraction, sequencing and analysis}

PBMCs were isolated from the blood of RA patients and healthy donors via
centrifugation over Ficoll-Paque Plus (GE Healthcare, Sweden). RNA was
extracted from PBMCs using Trizol: chloroform, precipitated in
isopropanol. Sample quality was assessed using Nanodrop. Total RNA was
enriched for mRNA using poly-A selection. In brief, mRNA was fragmented,
reverse transcribed, adapted with sequencing primers and sample
barcodes, size selected, and PCR enriched. Libraries were sequenced on
the HiSeq 2000 platform. RNA sequence reads were aligned to the human
reference genome (NCBI, hg19) using TopHat2 aligners. Normalised gene
counts were calculated using HTSeq, and differential gene expression
between samples was identified via edgeR. Gene expression (RPKM) values
were calculated using Cufflinks v2.0.2. Gene Ontology
(http://geneontology.org/) associations were determined using Gorilla.
Network construction was based on the Ingenuity Pathway Analysis
(Qiagen, CA, USA) experimental evidence database.

\textbf{Cell culture}

PBMCs were isolated from the blood of RA patients as described above.
CD14+ monocytes were positively selected via magnetic-activated cell
sorting (Miltenyi Biotec, Germany) and an anti-CD14 antibody. The
isolated CD14+ monocytes (1.5 \selectlanguage{ngerman}× 10\textsuperscript{5} cells per well in
48-well plates or 6 × 10\textsuperscript{5} cells per well in 12-well
plates) were incubated in RPMI-1640 media containing 10\% fetal bovine
serum (FBS). Osteoclast differentiation was induced from CD14+ monocytes
treated with macrophage colony-stimulating factor (M-CSF, 50 ng/ml) and
receptor activator of nuclear factor kappa-\selectlanguage{greek}Β \selectlanguage{english}ligand (RANKL, 100 ng/ml).
C-X-C motif chemokine ligand 2 (CXCL2, 100 ng/ml or otherwise as
indicated) were added to the cultures. The medium was replaced every 3
days.

RAW264.7 macrophages (1.0 \selectlanguage{ngerman}× 10\textsuperscript{4} cells per well in
48-well plates or 4.5 × 10\textsuperscript{4} cells per well in 12-well
plates) were incubated in DMEM media containing 10\% FBS. Osteoclast
differentiation was induced by M-CSF (10 ng/ml) and RANKL (50 ng/ml)
with or without CXCL2.

\textbf{Flow cytometry}

To identify the expression of C-X-C motif chemokine receptor 2 (CXCR2),
CD14+ monocytes were incubated for 45 min at 4°C with anti-human CXCR2
in the dark. Cells were washed twice with staining wash buffer and
centrifuged at 1000 rpm for 5 minutes to pellet the cells. Stained cells
were resuspended in 200 \selectlanguage{greek}µ\selectlanguage{english}l of PBS then analyzed via flow cytometry. In
each case, staining was compared with that of the appropriately labeled
isotype control antibody.

\textbf{Cell migration}

Monocyte migration testing was performed with 5\selectlanguage{greek}μ\selectlanguage{english}m pore-size
polycarbonate membrane transwell tissue culture inserts (Corning, USA).
Cells (5 \selectlanguage{ngerman}× 10\textsuperscript{4} per well) were seeded into the wells in
the top chamber in 10\% FBS medium, and CXCL2 was added to the lower
chamber in 10\% FBS medium. After incubation for 1 hour at 37°C the
medium was removed and the filters were fixed with 4\% paraformaldehyde
for 20 minutes. The filters were then stained with 0.1\% crystal violet
for 15 minutes. The number of cells that had migrated from the upper
surface of the filter to the lower surface of the filter was counted.

\textbf{Quantitative PCR}

Total RNA was extracted using TRIzol reagent (Invitrogen), and 1 \selectlanguage{greek}μ\selectlanguage{english}g of
total RNA was reverse-transcribed into cDNA using a FastQuant RT Kit
(with gDNase) (Tiangen Biotech). RT-PCR was carried out using the
QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific) with
Talent qPCR PreMix (SYBR Green) (Tiangen Biotech). Data were normalized
to the expression of GAPDH. The primer nucleotide sequences for PCR were
obtained from GenBank database and synthesized by Sangon Biotech
(China). The primer sequence information used in the study was shown in
the supplementary material, Table1.

\textbf{Enzyme-linked immunosorbent assay}

CXCL2 levels in sera from ACPA+ RA, ACPA- RA and healthy controls, as
well as levels in the supernatants of CD14+ monocyte cultures were
measured using human CXCL2 enzyme-linked immunosorbent assay kits in
accordance with the manufacturer's instructions.

\textbf{TRAP staining}

TRAP staining of CD14+ monocytes and RAW264.7 macrophages was performed
after culturing with or without CXCL2 in the presence of M-CSF and
RANKL. TRAP staining was conducted using the TRAP kit (387A-1KT, Sigma)
in accordance with the manufacturer's instructions. TRAP-positive
multinucleated cells containing more than 3 nuclei were identified as
osteoclasts and counted under a microscope.

\textbf{F-actin ring immunofluorescence}

Cells were fixed with 4\% paraformaldehyde and incubated with
FITC-conjugated anti-actin antibody for 1 hour at 25\selectlanguage{ngerman}°C. After a PBS wash
the cells were incubated with DAPI for 5 minutes at 25°C, then analyzed
using an Olympus BX51 microscope (Tokyo, Japan).

\textbf{Scanning electron microscopy}

Cells on dentine were fixed in 4\% glutaraldehyde, dehydrated via graded
alcohol solutions then graded (50\% to 100\%) hexamethyldisilazane
solutions (Sigma-Aldrich, MO, USA), then air-dried. Dentine slices were
then mounted onto aluminium stubs (EMS, Hatfield, PA, USA), sputtered
with gold, and examined using a scanning electron microscope (Philips
SEM 505, Best, Netherlands).

\textbf{Bone resorption assay}

Functional evidence of osteoclast formation was determined via a lacunar
resorption assay system using cell cultures on dentine slices as
previously described(17). Cells were removed from the dentine slices by
treatment with 0.1 M ammonium hydroxide. The dentine slices were washed
in distilled water and ultrasonicated to remove adherent cells, then
stained with 0.5\% (v/v) toluidine blue to reveal areas of lacunar
resorption, and examined via light microscopy.

\textbf{Western blotting}

Total protein was extracted using RIPA lysis buffer (Applygen, Beijing,
China). A total of 40 \selectlanguage{greek}μ\selectlanguage{english}g of protein was loaded into a 10\% sodium
dodecyl sulphate-polyacrylamide gel and electrophoretically transferred
to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA,
USA). After blocking with 5\% milk for 1 h, the membranes were incubated
with specific antibodies at the indicated dilutions for 12--16 h at 4\selectlanguage{ngerman}°C.
The membranes were scanned using an Odyssey Sa Imaging System (LICOR
Biosciences, Lincoln, NE, USA). The antibody information used in this
study was shown in the supplementary material, Table 2.

\textbf{Statistical analysis}

Statistical analysis of RNA sequencing count data was conducted using
edgeR with a 5\% FDR. All experiments were repeated at least three times
and are presented as the mean±standard deviation and the median with
interquartile range. Statistical analysis were assessed via Spearman
correlation, Mann-Whitney test and one-way analysis of variance followed
by Tukey's test for multiple comparisons. p values \textless{} 0.05 were
considered statistically significant.

\textbf{Results}

\textbf{Selection of candidate genes using RNA sequencing}

Further analysis was conducted on RNA sequencing data from 7 samples; 2
ACPA+ RA patients, 2 ACPA- RA patients and 3 healthy controls.
Differentially expressed genes (DEGs) were analyzed in the two different
sample types, and three groups of DEGs were identified (ACPA+ vs.
control, ACPA+ vs. ACPA-, ACPA- vs. control). We identified 1007 DEGs
(522 up-regulated and 485 down-regulated) between ACPA+ and control, 628
DEGs (344 up-regulated and 284 down-regulated) between ACPA+ and ACPA-,
and 1097 DEGs (549 up-regulated and 548 down-regulated) between ACPA-
and control (Fig. 1A and 1B). All these DEGs exhibited distinct
expression patterns in the three sample types (Fig. 1C).

Pathway enrichment analysis/ingenuity pathway analysis of DEGs yielded
19 canonical pathways that were significantly enriched between ACPA+ and
control, 24 between ACPA+ and ACPA-, and 35 between ACPA- and control
(see supplementary information). Some of these pathways were enriched in
more than one comparison (Fig. 1D). The agranulocyte adhesion and
diapedesis pathway was particularly enriched in ACPA+ vs. control and
ACPA+ vs. ACPA- (Fig. 1D). Different DEGs were involved in the
agranulocyte adhesion and diapedesis pathway in two comparisons (Fig. 1E
and 1F), and of these, CXCL2 and C-X-C motif chemokine ligand 7 (CXCL7)
were exclusively up-regulated in ACPA+ samples (Fig. 1E).

\textbf{Validation of candidate genes expression in ACPA+ RA patients}

To validate the differential expression levels of candidate genes in
ACPA+ and ACPA- RA patients, the expression levels of CXCL2 and CXCL7
were measured using real time PCR. CXCL2 levels were higher in ACPA+ RA
patients (n=40) than in ACPA- RA patients (n=40) and healthy controls
(n=40) (Fig. 2A), confirming that CXCL2 was differentially expressed in
ACPA+ RA patients and ACPA- RA patients. However, there was no
significant difference in CXCL7 expression between ACPA+ RA patients and
ACPA- RA patients.

\textbf{CXCL2 levels in serum and CD14+ cell supernatant}

CXCL2 levels in sera were detected in 70 ACPA+ RA patients, 37 ACPA- RA
patients and 40 healthy controls. Age and sex were balanced between the
groups, whereas the disease duration, RF positive rate and bone erosion
positive rate were significantly different among ACPA+ RA patients and
ACPA- RA patients (Table 1).

Median serum CXCL2 in RA patients (1083.43, (586.94,1569.50) pg/ml) was
significantly higher than in healthy controls (301.44, (217.62,422.26)
pg/ml) (p \textless{} 0.001; Fig, 2B). And median serum CXCL2 was
significantly higher in ACPA+ RA patients than in ACPA- RA patients (p
\textless{} 0.05; Fig. 2C). Correlation analysis showed that CXCL2 was
positively correlated with DAS28 (r=0.4594, p \textless{} 0.001), ESR
(r=0.3985, p \textless{}0.001) and CRP (r=0.3726, p \textless{} 0.001)
(Fig. 2D). However, the CXCL2 level was not related to disease duration
(r=-0.084, p=0.391) and RF titer (r=0.1358, p=0.309). Serum CXCL2 was
significantly higher in RA patients with bone erosion than in RA
patients without bone erosion (p \textless{} 0.05; Fig. 2E).

CXCL2 expression was significantly higher in the supernatant of CD14+
monocyte cultures derived from ACPA+ RA patients than from the ACPA- RA
patients (Fig. 2F).

\textbf{CXCL2 promoted migration of CD14+ monocytes from RA patients}

Flow cytometry analysis using anti-CXCR2 antibody revealed that CD14+
monocytes from ACPA+ patients expressed CXCR2 at a higher level than
those from ACPA- patients, but the difference was not statistically
significant (Fig. 3A). In the transwell experiments, CXCL2 significantly
increased the number of migrating monocytes in cultures derived from RA
patients (Fig. 3B).

\textbf{CXCL2 increased osteoclast differentiation and enhanced
osteoclastic bone resorption}

In experiments conducted to evaluate the effects of CXCL2 on
osteoclastogenesis, cultures from all groups formed osteoclasts on
plastic and the average numbers of osteoclasts were similar in the
control cultures and cultures exposed to a low concentration of CXCL2
(10 ng/mL). However, high doses of CXCL2 tended to promote the formation
of osteoclasts (Fig. 4A). Formation of the F-actin ring by osteoclasts
is a necessary step in bone resorption. Immunofluorescence microscopy of
CD14+ monocytes cultured for 14 days treated with CXCL2 (100 ng/ml) in
the presence of M-CSF and RANKL revealed that F-actin rings were
significantly increased in number (Fig. 4B). CXCL2 stimulation also
increased cell fusion, resulting in multinucleated cells (Fig. 4C),
which suggested that CXCL2 promoted osteoclast formation.

Functional evidence of osteoclast formation was obtained via the lacunar
resorption assay system using cell cultures on dentine slices. The
result showed that the resorption area was increased in bone slices
exposed to CXCL2 (Fig. 4D). Expression of the osteoclast markers RANK,
cathepsin K, and TRAP was significantly increased after 3 days of
stimulation with the CXCL2 (100 ng/mL) in the presence of M-CSF (50
ng/ml) and RANKL (100 ng/ml) (p \textless{} 0.05; Fig. 4E).

Consistent with the results obtained in CD14+ monocytes, the effects of
exogenous CXCL2 on osteoclast formation were also observed in RAW264.7
cells. Compared with the control group, much larger multinucleated cells
were evident in the CXCL2 groups (Fig. 5A). Expression of osteoclast
markers such as cathepsin K and TRAP was also significantly increased in
the CXCL2 group after 3 days of stimulation in the presence of M-CSF and
RANKL (p \textless{} 0.05; Fig. 5B). These results suggest that CXCL2
enhances osteoclast formation and bone resorption. In addition, CXCL2
significantly increased the expression of NFATc1 and c-Fos by RAW264.7
cells after 5 days of stimulation with M-CSF (10 ng/ml) and RANKL (50
ng/ml) (p \textless{} 0.05; Fig. 5C).

\selectlanguage{greek}\textbf{CXCL2 stimulated osteoclastogenesis via ERK MAPK and NFκB
pathways}\selectlanguage{english}

To explore the signal pathways involved in the osteoclastogenesis with
the stimulation of CXCL2, RAW264.7 cells were incubated in
osteoclast-promoting medium with or without CXCL2, western blotting at
different time-points was conducted to detect the phosphorylation of
I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α, \selectlanguage{english}p65, ERK1/2 and JNK. The addition of CXCL2 dramatically promoted
phosphorylation of p65 and ERK1/2 (Fig. 6A, B), with no effect on the
phosphorylation of I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}or JNK (Fig. 6C, D).

\textbf{Discussion}

RNA sequencing technology has been used to detect differences in gene
expression profiles in RA patients for the last few years. Several gene
expression profiling studies of synovial tissues and PBMCs from RA
patients have revealed marked variation in gene expression profiles that
have facilitated the identification of distinct molecular disease
mechanisms involved in RA pathology(18-21). The heterogeneity of RA was
demonstrated by the presence of distinct autoantibody specificities such
as ACPA in serum, differential responsiveness to treatment, and
variability in clinical presentation. RA patients can be stratified into
two subgroups defined by the presence or absence of ACPA, and ACPA+
patients exhibit more severe inflammation and radiographic damage than
ACPA- patients(5-7). In the present study, various genes were compared
in ACPA+ and ACPA- patients via RNA sequencing, and two significantly
increased chemokines, CXCL2 and CXCL7 were identified in ACPA+ patients.
The next more focused analysis using PCR technology on many more
patients found that only CXCL2 was differentially expressed in ACPA+ RA
patients and ACPA-RA patients. There was no significant difference in
CXCL7 expression between ACPA+ RA patients and ACPA- RA patients.

CXCL2 was first identified as a major chemokine produced by
endotoxin-treated macrophages(22), which bind to the G-protein coupled
receptor CXCR2 expressed on macrophages, neutrophils, and epithelial
cells(23). In previous studies, CXCL2 level was found to be higher
expressed in RA patients than in normal controls(24). Xiaokun \emph{et
al.} downloaded microarray datasets of GSE1919, GSE12021, and GSE21959
(35 RA samples and 32 normal controls) from the Gene Expression Omnibus
database (https://www.ncbi.nlm.nih.gov/geo/) and identified DEGs in RA
samples. They found that CXCL2 was strongly associated with DEGs
involved in RA progression(24). Jacobs \emph{et al.} (25) performed a
microarray analysis to characterize the molecular events underlying
pathology in autoantibody-mediated arthritis and reported that CXCL2 was
up-regulated in parallel with the disease. Jeongim \emph{et al.} (26)
reported that CXCL2 was significantly higher in synovial fluid and sera
from RA patients compared with corresponding samples from osteoarthritis
patients. In the present study, serum CXCL2 was significantly increased
in RA patients compared with healthy controls, and serum CXCL2 was
higher in ACPA+ RA patients than in ACPA- RA patients. Compared with
ACPA- patients, ACPA+ patients had longer disease duration and higher
positive rate of RF. To exclude the impact of these two factors on CXCL2
level, correlation analysis was applied and showed that CXCL2 was
irrelevant to disease duration and RF titer.

CXCL2 was involved in various biological progresses, such as
angiogenesis, inflammation and cancer biological behaviors(23, 27-30).
CXCL2 was also considered to be a proinflammatory factor in RA(24). In
line with this, our study showed that CXCL2 was positively correlated
with DAS28, ESR and CRP. Moreover, recent studies revealed CXCL2 was
also involved in the process of osteoclastogenesis(26, 31). In the
present study, we also found serum CXCL2 was significantly higher in RA
patients with bone erosion than in RA patients without bone erosion.
Thus, we hypothesized that CXCL2 could be one of the key molecules
upregulated in RA progression, especially in ACPA+ RA which exhibit more
radiographic damage, and focused on the CXCL2 for subsequent analyses.

As the most important osteoclast precursors, CD14+ monocytes were
derived from ACPA+ patients and exhibited elevated CXCL2 secretion
compared with those derived from ACPA- patients. CXCR2 is known to be
the major receptor for CXCL2(32). It has been suggested that CXCL2 acts
as a chemoattractant for various types of cells by binding to CXCR2, and
monocytes constitutively express CXCR2(32). However, whether there is a
difference in the expression of cell surface CXCR2 in CD14+ monocytes
from ACPA+ and ACPA- RA patients remains unclear. In the present study,
we found that CXCR2 expression was higher on the surfaces of CD14+
monocytes derived from ACPA+ patients than those from ACPA- patients,
but the difference was not statistically significant.

ACPA+ RA patients usually develop more severe radiological damage, and
enhanced osteoclastogenesis may be involved in the bone erosion(7).
Osteoclasts are polykaryocytes formed via the fusion of mononuclear
monocytic precursors such as monocytes in peripheral blood. We surmise
that elevated CXCL2 may recruit more monocytes to joint sites and
augment the formation of osteoclasts. The present study showed that
CXCL2 promoted the migration, differentiation, and function of
osteoclasts in experiments using CD14+ monocytes isolated from RA
patients.

The effects of CXCL2 on activating the signaling pathway during
osteoclast differentiation were examined to explore the molecular
mechanisms of the observed enhancement effect on osteoclastogenesis. The
addition of CXCL2 resulted in dramatically increased phosphorylation of
p65 and ERK1/2, while it did not affect the phosphorylation of I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}or
JNK. NFATc1 and c-Fos are critical transcription factors in the
regulation of osteoclast differentiation(33). In the current study,
NFATc1 and c-fos were increased significantly with stimulation of CXCL2
in the presence of M-CSF and RANKL. NFATc1 is an important regulatory
factor in the process of osteoclast differentiation mediated by
RANKL-activated MAPK and NF\selectlanguage{greek}κ\selectlanguage{english}B signalling pathways(34). Therefore, it
suggests that CXCL2 may promote osteoclast differentiation and bone
resorption by regulating NFATc1 expression via interfering with the
phosphorylation of NF\selectlanguage{greek}κ\selectlanguage{english}B p65 and MAPK ERK1/2.

Jeongim \emph{et al.} (26) reported that RANKL significantly increased
the expression of CXCL2 in bone marrow-derived macrophages (BMMs) and
that CXCL2 mediated RANKL-dependent differentiation of osteoclasts from
BMMs in mice. The osteoclastogenesis-enhancing effects of CXCL2 were
further corroborated by their investigation with an in vivo bone
resorption model. Notably, CXCL2 alone induced significant bone loss in
mice calvarial similar to that induced by RANKL. They also reported that
CXCL2 mediated lipopolysaccharide (LPS)-induced osteoclastogenesis in
RANKL-primed precursors (i.e. BMMs) (31). LPS stimulated the production
of CXCL2 in BMMs, and the conditioned medium from LPS-treated BMMs could
enhance the migration of osteoclast precursors, which was blocked by
treatment with CXCL2-neutralising antibody or CXCR2 receptor antagonist.
Blocking CXCL2 also reduced LPS-induced osteoclastogenesis, and in
addition, CXCL2 neutralization prevented bone destruction in mice
treated with LPS, suggesting a critical role of CXCL2 in the process of
osteoclastogenesis. In the present study, CXCL2 was also evidently
involved in the process of osteoclast differentiation in RA patients.

Therapies targeting CXCL2/CXCR2 have been tested in animal models of
arthritis with concomitant reduction in neutrophil recruitment and tumor
necrosis factor-\selectlanguage{greek}α \selectlanguage{english}production(35, 36), and immunization against CXCL2 was
reportedly efficient in delaying the onset of arthritis and reducing
disease severity in a murine collagen-induced arthritis model(37). The
novel orally-active non-competitive allosteric inhibitor of CXCL2 known
as DF 2162 significantly ameliorates AIA in rats, an effect that is
quantitatively and qualitatively similar to that of anti-tumor necrosis
factor antibody treatment(35). In addition, the CXCR2 antagonist
SCH563705 led to a dose-dependent decrease in clinical disease scores
and paw thickness measurements, and clearly reduced inflammation and
bone and cartilage degradation in a mouse model of arthritis(36). The
results of our study imply that targeting CXCL2/CXCR2 as a strategy to
treat RA may contribute to protection from bone destruction by directly
inhibiting bone resorption, in addition to the already suggested
anti-inflammatory effects.

In conclusion, we identified novel pathways associated with ACPA+ RA
patients using RNA sequencing, and detected higher CXCL2 expression in
ACPA+ RA patients than in ACPA- RA patients. Increased levels of CXCL2
led to NF\selectlanguage{greek}κ\selectlanguage{english}B activation in CD14+ monocytes from RA patients during the
osteoclastogenic process. These results suggest a previously unreported
role of CXCL2 during osteoclastogenesis in RA patients, and indicate
that CXCL2 blockade might be a novel therapeutic strategy in RA.

\textbf{Supplementary data}

Supplementary data are available online.

\textbf{Acknowledgements}

We gratefully thank the Medical Research Center of Peking University
Third Hospital for providing experimental equipment and technical
support. This work was supported by the National Natural Science
Foundation of China (No.81571573 and No. 81771744).

\textbf{Authors' contributions}

LS, XW, NH, ZA, RY, CL, YL, YL and XL carried out the molecular
biochemical studies. LS, XW and NH drafted the manuscript and performed
the statistical analysis. XF and JZ participated in the design of the
study and helped to draft manuscript. All authors read and approved the
final manuscript.

\textbf{Competing interests}

The authors declare no conflicts of interest.

\textbf{Table 1 Baseline characteristics of the enrolled RA patients}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Total (n=107)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
ACPA + (n=70)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
ACPA -- (n=37)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
P Value\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Age (mean year)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
55.94\selectlanguage{ngerman}±14.11\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
55.13\selectlanguage{ngerman}±13.64\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
57.49\selectlanguage{ngerman}±15.02\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.414\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Gender (male/female)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
26/81\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
16/54\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
10/27\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.632\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Duration (months)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
24 (12,108)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
60 (17,183)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
13 (5,36)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{0.000}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
RF-positive\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
57.94\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
82.86\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
10.81\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{0.000}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
ESR (mm/h)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
32 (14,60)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
33 (14.5,60)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
29 (13.5,57.5)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.692\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CRP (mg/dl)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.86 (0.51,5.22)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.565 (0.5,4.37)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2.46 (0.61,7.72)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.250\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
DAS28\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
5.84 (4.41,7.59)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
6.62 (4.13,7.77)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
5.64 (4.5,6.71)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.187\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Bone erosion\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
28.95\% (n=22/76)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
38\% (n=19/50)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11.54\% (n=3/26)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{0.016}\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Data are represented as mean \selectlanguage{ngerman}± SD and median with interquartile range.

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\textbf{Hosted file}

\verb`fig 20200405.docx`  available at \url{https://authorea.com/users/314148/articles/444535-increased-expression-of-cxcl2-in-acpa-positive-rheumatoid-arthritis-and-its-role-in-osteoclastogenesis}

\selectlanguage{english}
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\end{document}

