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 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 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 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 IκBα 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 NFκB 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 NFκB p65 and MAPK ERK1/2.
Jeongim 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-α 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 NFκB 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.