Extracellular vesicles are associated with the systemic inflammation of patients with seropositive rheumatoid arthritis
Extracellular vesicles are associated with the systemic inflammation of patients with seropositive rheumatoid arthritis
Luis A. Correa
OPEN Patients with rheumatoid arthritis (RA) and autoantibodies, such as rheumatoid factor and those against cyclic citrullinated peptides, are designated as seropositive and have a more severe disease with worse prognosis than seronegative RA patients. Understanding the factors that participate in systemic inflammation, in addition to articular commitment, would allow better treatment approaches for prevention of RA comorbidities and disease reactivation. We evaluated whether monocyte subsets and extracellular vesicles (EVs) could contribute to this phenomenon. Seropositive patients had higher levels of proinflammatory cytokines than those of seronegative patients and healthy controls (HCs); however, this systemic inflammatory profile was unrelated to disease activity. High frequencies of circulating EVs positive for IgG, IgM, CD41a, and citrulline, together with altered counts and receptor expression of intermediate monocytes, were associated with systemic inflammation in seropositive patients; these alterations were not observed in seronegative patients, which seem to be more similar to HCs. Additionally, the EVs from seropositive patients were able to activate mononuclear phagocytes in vitro, and induced proinflammatory cytokines that were comparable to the inflammatory response observed at the systemic level in seropositive RA patients; therefore, all of these factors may contribute to the greater disease severity that has been described in these patients.
Rheumatoid arthritis (RA) is characterized by a systemic inflammatory state that mainly affects joints but also
other organs, such as skin, eyes, lung, and the cardiovascular system1. Two groups of patients with RA are clearly
differentiated: the seropositive and seronegative type. Rheumatoid factor (RF), and antibodies against cyclic
citrullinated peptides (anti-CCP) are hallmarks of patients with seropositive RA2. The presence of anti-CCP in
patients with seropositive RA has been associated with more persistent inflammation, greater erosive joint
damage, more severe clinical manifestations and worse prognosis than those in patients with seronegative RA3. The
participation of RF in the disease?s development is not clear yet; however, it is proposed that this autoantibody
potentiates the pathological effects of anti-CCP4. After antigen recognition by these autoantibodies, the immune
complexes (ICs) formed can activate mononuclear phagocytes in circulation and joints, promoting the secretion
of inflammatory mediators, such as TNF-?, IL-6, and IL-1?5. These cytokines are biomarkers of systemic
inflammation in RA and contribute to the immunopathology of this disease because their blocking is associated with
clinical improvement and remission6?8.
The understanding of which circulating factors contribute to the systemic inflammation in RA is essential to
find more promising alternatives for the intervention and treatment of these patients. A high number of
extracellular vesicles (EVs) have been detected in circulation and synovial fluid of RA patients9,10. The role of EVs
in the pathogenesis of RA has begun to be elucidated; for example, these vesicles express autoantigens, such as
citrullinated peptides (CPs), form ICs, and participate in the delivery of miRNA, and fibroblast-like synoviocytes
activation11. EVs contribute in cell-to-cell communication and have a broad spectrum of effects on immune
responses, mainly on monocytes and macrophages, and induce TNF-?, IL-6, and IL-1? production11,12.
Different cell components are involved in the development of inflammation, including neutrophils,
mastocytes, T and B lymphocytes, and monocytes/macrophages. Activation of these cells leads to the production of
cytokines and mediators responsible for inflammation. More specifically, monocytes/macrophages have been
found to be activated in RA13?15, to massively infiltrate synovial membranes in RA16,17, and to produce TNF-?17.
It has been shown in RA patients that circulating EVs forming ICs (EV-ICs)10,18 induce leukotriene production
by neutrophils in vitro, which indicates that these vesicles may contribute to the inflammatory process. We
propose that EVs can activate monocytes in circulation and contribute to the systemic inflammatory process in RA,
similar to that observed in other systemic autoimmune diseases19. However, the effect of EVs on monocytes has
not been previously associated with RA. Therefore, we evaluated whether patients with seropositive RA had any
particular profile of circulating EVs and monocytes that could be associated with their systemic inflammatory
response and assessed the direct effect of EVs and EV-ICs in mononuclear phagocytes.
Systemic inflammatory response in seropositive patients is characterized by high serum levels
of TNF-?, IL-6, and IL-1?. Alterations in serum cytokine levels have been described in different systemic
inflammatory processes. Serum cytokines were measured as indicators of the systemic inflammatory state of RA
patients. As shown in Fig.?1A, the classification of patients according to disease activity by disease activity score
(DAS)?28 showed great variability in cytokine concentrations; patients classified under remission or low
activity had either elevated or very low serum concentrations of cytokines. However, when patients were classified
according to seropositivity, the groups of anti-CCP+RF+/? and anti-CCPhiRFhi (seropositive) patients had
significantly elevated levels of TNF-?, IL-6, IL12p70, and IL-1?, but not IL-8, relative to those in healthy controls (HCs);
anti-CCP?RF? (seronegative) patients did not show such increases (Fig.?1A). Interestingly, when we evaluated the
cytokines levels in patients with other systemic autoimmune disease (SADs), such as systemic lupus
erythematosus (SLE), we did not find differences in proinflammatory cytokines serum concentrations compared with HCs
(Data not shown). These findings indicate that serum cytokine concentrations are not increased in all SADs and
may be characteristic of seropositive RA patients (anti-CCP+RF+/? and anti-CCPhiRFhi).
A principal component analysis (PCA) was performed to define associations among serum cytokines (IL-1?,
IL-12p70, IL-6, TNF-?, and IL-10), C-reactive protein (CRP), and DAS28 variables in patients with seropositive
and seronegative RA and HCs (Fig.?1B). The PCA showed two components that explained the 65% of the
variability and enabled separation of the variables between the two groups: a group associated with the
proinflammatory cytokines that included most of the seropositive patients and a group associated with CRP, IL-10, and the
DAS28 that was not clearly associated with seropositivity/seronegativity. HCs and anti-CCP?RF? patients were
not defined on the basis of these variables in either group and remained together. With the eigenvalues obtained
from the PCA of each variable, a heat map was created. The heat map showed that the cytokines IL-1?, IL-12p70,
TNF-?, and IL-6 defined seropositive patients better than did the DAS28 and CRP (Fig.?1B).
High counts of intermediate monocytes in patients with seropositive RA. Alterations in the
frequency of circulating monocyte subsets20 and activation of total monocytes producing proinflammatory
cytokines13?15 have been described in patients with RA. Therefore, we evaluated if the number, frequency, and
phenotype of monocyte subsets were associated with seropositivity of patients with RA. A decrease in the
proportion of classical monocytes was observed in anti-CCPhiRFhi patients, which was not reflected in absolute counts
(Fig.?2A,B). The intermediate monocytes were significantly elevated in both proportion and counts in the
seropositive patients relative to HCs. Additionally, the non-classical monocytes were reduced in both the proportion
and number in anti-CCPhiRFhi patients (Fig.?2A,B). The expressions of receptors associated with the recognition
of EVs and migration of monocyte subsets were evaluated in intermediate monocytes because these cells were
the most affected in count and frequency in seropositive patients. Low expressions of HLA-DR and CX3CR1 in
seropositive patients, and low expressions of CD86, CD36, CCR2, and CCR5 in anti-CCPhiRFhi patients were
observed compared with HCs (Fig.?2C). Low expression of CD18 was found in all monocyte subsets in
seronegative patients relative to that in anti-CCPhiRFhi patients and HCs (data not shown).
EVs of seropositive patients are platelet-derived, CPs+, and form ICs. Recent reports indicate that
EVs have a pivotal role in autoimmune diseases21,22 because of different pleiotropic effects on mononuclear
phagocytes and other components of the immune system21. All patients with RA seem to display elevated EV counts;
however, only anti-CCP+RF+/? patients showed a significant difference compared with HCs (Fig.?3A). Regarding
EV-size distribution, anti-CCP?RF? and anti-CCP+RF+/? patients had significantly decreased proportions of
0.1?1.0-? m EVs and elevated proportions of 1?3.0-? m and 3?6.0-? m EVs (Fig.?3A,B). The EV phenotype was
also studied to identify the cellular source. The seropositive patients had elevated proportions of EV-CD41a+,
and the anti-CCP?RF? group had elevated EV-CD105+ relative to that of the anti-CCPhiRFhi patients (Fig.?3C).
Taking into account that approximately 50% of the EVs were derived from leukocytes (EV-CD45+), we
evaluated different leukocyte sources. All groups of patients had elevated frequencies of EV-HLA-DR+, and the
frequency of EV-CD14+ appeared to be decreased, but the difference was statistically significant only in the
anti-CCP+RF+/? group (Fig.?3C).
In addition to EV cell sources, components of EVs involved in the RA physiopathology were evaluated.
The EV-ICs (IgG+ IgM-, IgG-IgM+, and IgG + IgM+) and EV-CPs+ were significantly elevated in
seropositive patients relative to those in HCs. The anti-CCP+RF+/? patients had higher frequencies of EV-C1q+ and
EV-High-mobility group box 1 (HMGB1)+ than those in the other groups; the EVs of seronegative patients
were similar to those of HCs (Fig.?3D). The cell sources of EV-ICs, EV-CPs+, EV-C1q+, and EV-HMGB1+ were
both leukocytes and platelets in total patients with RA; however, platelets were the main source in seropositive
patients (data not shown). To illustrate the variations in ICs content of the EVs, 5 representative individuals
of each group of patients with RA and HCs were randomly selected and a t-Distributed Stochastic Neighbor
Embedding (t-SNE) analysis was performed. As expected, it was observed that vesicles from patients with
seropositive RA had higher frequencies and a wider distribution of EV-IgM+ and EV-IgG+ than those of HCs and
seronegative patients (Fig.?3E).
EVs and monocyte subsets from synovial fluid and circulation share some characteristics. To
evaluate whether the phenotype of EVs and monocyte subsets observed in circulation of seropositive patients also
can be found in the synovial fluid (SF) of these patients, the SF of one anti-CCP+RF+/? and two anti-CCPhiRFhi
patients were assessed. Approximately 20% of the SF infiltrating cells corresponded to monocytes, and strikingly,
70% of these cells corresponded to intermediate monocytes (Fig.?4A). However, there was a greater expression
per cell of HLA-DR, CD86, CCR2, CX3CR1, CCR5, and CD32 but lower expression per cell of CD36, CD11b,
and Fc?R in intermediate monocytes of SF than those in circulating cells of the same patients (Fig.?4B and data
not shown). Interestingly, mononuclear phagocytes from patients with seropositive RA of a 24 h culture showed
endothelium (CD205), erythrocytes (CD235a), and other sources (EV negative for the evaluated molecules)
from anti-CCP?RF?, anti-CCP+RF+/?, and anti-CCPhiRFhi RA patients and HCs. Right: frequency of
circulating EVs from different leukocyte sources in anti-CCP?RF?, anti-CCP+RF+/?, and anti-CCPhiRFhi RA
patients and HCs. (D) Frequency of circulating EVs positive to IgG + IgM-, IgG-IgM+, IgG+IgM+, C1q+,
HMGB1+, and CP+ from anti-CCP?RF?, anti-CCP+RF+/?, and anti-CCPhiRFhi RA patients and HCs.
Comparisons among the study groups were made by performing the Kruskal?Wallis test and Dunn?s post-hoc
test. (E) Collective t-SNE of EVs (1 ? 105) derived from five samples from each group analyzed and plotted
(n = 20). Every dot represents a single EV, and the color indicates ArcSinh5-transformed expression values for
each given marker analyzed and calculated over EVs from all samples varying from blue for lower expression to
red for higher expression.
higher expression of CD32 and lesser expressions of CD11b, CD36, and Fc?R than those in HCs (Supplementary
Figure?1?S1). Then, the phagocytes observed in the SF of patients could acquire this phenotype once they begin
their process of tissue differentiation23.
Regarding EVs of SF and similar to circulating EVs, platelets and leukocytes were the main sources of these
vesicles; also, the EVs of greater size (FSC-A) and complexity (SSC-A) were those that mainly form ICs in SF;
these EVs also contain HMGB1, HLA-DR, and CP in different proportions (Fig.?4C).
EVs, intermediate monocytes, and proinflammatory cytokines allow the discrimination of
patients with RA according to seropositivity. To establish which of the variables evaluated in
monocyte subsets and EVs from blood are associated with autoantibody levels and systemic inflammation in RA
patients, a discriminant analysis was performed with samples of patients and HCs. Twenty-three predictor
variables that included intermediate monocyte counts and receptors (CD16, CD64, CD32, CD86, HLA-DR, CCR2,
CCR5, CX3CR1), CD18 of three monocyte subsets, cytokines (IL-6, IL1?, and TNF-?), and frequencies of EVs
positive for IgG, IgM, HMGB1, CPs, CD41a, and HLA-DR were included. The four study groups were
significantly discriminated by these variables (p < 0.05), with a confidence level of 95%. However, anti-CCPhiRFhi and
anti-CCP+RF+/? patients showed slight overlap. Interestingly, although HCs and anti-CCP?RF? segregated to
the same side, they were mutually excluded (Fig.?5A), which suggested that EVs and intermediate monocytes are
closely associated with the systemic inflammation observed in seropositive patients.
EVs from seropositive patients induce proinflammatory cytokine production by mononuclear
phagocytes. To evaluate whether EVs from seropositive patients induce production of the cytokines related
to the systemic inflammation detected in seropositive patients, we first evaluated the binding and
internalization of these vesicles. Intermediate monocytes and mononuclear phagocytes of a 24 h culture from RA patients
bound and internalized higher levels of EV-ICs than those of un-opsonized EVs (Fig.?5B). Finally, we found that
in response to EVs, mainly EV-ICs, there were accumulations of IL-1?, IL-6, and TNF-? in the supernatants of
mononuclear phagocytes of RA patients and HCs (Fig.?5C), which suggested that mononuclear phagocytes, after
their EVs interaction, could contribute to the systemic inflammation observed in seropositive patients.
Multiple studies have identified several circulating cytokines and chemokines that reflect the systemic
inflammation in RA24,25. However, little is known about the components responsible for this. We observed that higher levels
of the serum cytokines IL-1?, IL-12p70, IL-6, and TNF-? were characteristic of patients with seropositive RA; in
contrast, seronegative and SADs patients did not have elevated levels of these cytokines. The seropositive patients
also exhibited high frequencies of circulating EVs positive for CD41a, IgG, IgM, and CPs, and altered counts,
frequencies, and receptor expressions of monocyte subsets, mainly intermediate ones. These changes were not
observed in seronegative patients, who more closely resembled HCs. Actually, EV-ICs from seropositive patients,
more than EVs, induced the production of IL-1?, IL-6, and TNF-? by mononuclear phagocytes of RA patients
and HCs in vitro. No one of the alterations observed in intermediate monocytes and EVs from RA patients were
associated with disease activity evaluated by DAS28 (Figure?S2 and data not shown). Therefore, these results
together with the discriminant analysis suggest that in RA, the seropositive patients are those who mainly present
systemic inflammation, which seems to be partially explained by the effect of EVs on mononuclear phagocytes. It
is currently believed that seropositive and seronegative are two different RA entities, each with its own
pathogenesis, disease course, and genes involved26. Our results agree with this hypothesis because seropositive patients, who
have more severe disease and worse outcomes27, exhibited evident systemic inflammation.
Different circulating components, such as cytokines, EVs, and monocyte subsets, were identified according to
the seropositivity status of RA patients. A few studies have analyzed IL-17, IL-1528, and IL-3329 cytokines
according to anti-CCP positivity. Other studies have focused on evaluating cytokines in SF; G?mez-Puerta et al. in
20133 found higher levels of cytokines IL-1?, IL-10, and IL-17 and the CCL20 chemokine in anti-CCP+ patients
than those in seronegative patients. However, it was not clear if there was an association between the local and
systemic inflammation process in those patients. Recent evidence has suggested the essential role of cytokines in
anti-citrullinated protein antibody (ACPA)-positive patients with RA. It has been observed that the levels of IL-6,
IL-17, and IL-8 were significantly elevated in SF from synovial ACPA-positive patients, and that the levels of IL-8
positively correlated with neutrophil counts and worse clinical manifestations in patients with RA30. Another
study showed that exposure of osteoclast to ACPAs leads to a preferential release of IL-8, which mediates an
autocrine activation of these cells31. Although we did not find significant differences in the serum levels of IL-8 among
seropositive patients versus HCs and seronegative patients, it is important to note that we evaluated the serum
and not SF concentrations of this cytokine. Therefore, it is possible that IL-8 participates in joint rather than in
systemic inflammation. Additionally, it is noteworthy that IL-8 has been more often related to the initial hit of
autoantibodies in RA, wherein this cytokine seems to be associated with the induction of initial bone resorption
and with higher neuropathic pain than with pannus formation and inflammatory pain32,33.
Our results suggest that systemic inflammatory process does not always coincide with articular inflammation,
as shown by the weak association between circulating cytokines and DAS28. This suggest that classifying the
disease as active based only on the articular commitment of DAS282 is insufficient to determine individuals with
systemic and non-joint inflammation. In addition, we observed that not all patients with high DAS28 exhibited
high levels of circulating inflammatory cytokines. This could be party explained by the generation of articular
pain unrelated to a systemic inflammatory process because the joints? sensory neurons can trigger pain in RA in
the absence of local inflammation, tissue damage, or infection34. Additionally, irreversible destructive joint lesions
often developed in the course of RA, remaining tender even after the inflammatory process subsides. Therefore,
DAS28 is an objective score that may lead to an overestimation of disease activity because it could be significantly
affected by the parameter of tender joint count. Clinically evaluating patients with RA based only in DAS28 may
therefore lead to an inaccurate reflection of the total disease activity (articular and systemic).
Previously, it has been shown that in vitro, EV-ICs caused marked upregulation of leukotriene-B4
production by neutrophils10; however, several studies have shown that monocyte/macrophage are the major source of
proinflammatory cytokines and chemokines in the inflamed RA joints, including TNF-? and IL-1?35. Here we
observed that EVs, particularly EV-ICs from RA patients, induced IL-6, IL-1?, and TNF-? production by
mononuclear phagocytes from HCs and RA patients. Although the inflammatory process in the joints could contribute
to circulating cytokines36, our data suggest that other circulating sources of these mediators seem to exist in
seropositive patients; particularly, our data support that EV-ICs and their interaction with monocytes could be
contributing to this phenomenon. However, it is still matter of investigation whether or not monocytes are the
main source of proinflammatory cytokines in seropositive patients at the circulation level.
Circulating cytokines and altered EVs are in a position to affect the function of distant tissues, including
adipose, skeletal muscle, liver, and vascular endothelium37,38. It has been shown that chronic elevation in cytokine
levels, irrespective of magnitude or cause, can generate a spectrum of proatherogenic changes that include insulin
resistance, dyslipidemia, prothrombotic effects, pro-oxidative stress, and endothelial dysfunction in RA patients25.
The ability to define the systemic inflammation in these patients could allow better establishment of the type and
dose of the treatment to prevent comorbidities associated with seropositive RA and/or avoid disease reactivation.
Therefore, the evaluation of serum cytokines, as well as some variables found in EVs and monocytes, could be a
promising tool for follow-up of patients. Additionally, the identification of systemic inflammation in RA patients
at the remission state defined by DAS28, could provide significant insights into the pathogenesis of RA and could
be used to guide the initiation of disease modifying drugs in these patients.
In agreement with previous studies9,10, we found that platelets constitute the most frequent source of EVs in
patients with RA; in addition, our study showed that this occurs mainly in seropositive patients. Furthermore,
these patients had the highest frequencies of circulating EV-ICs+ and EV-CPs+, which indicates that these
structures are an important source of ICs, autoantigens, and DAMPs in the circulation and SF of patients with RA, as
previously proposed by others10,18,21,22. Depletion of platelets, in a deficient collagen receptor glycoprotein-(GP)
VIa mouse model of RA, attenuated inflammatory arthritis, produced lower numbers of platelets-derived EVs
in vivo, with minor ankle thickness and poor bone and cartilage erosion relative to those in control mice9, which
suggested that these platelet-derived vesicles have an important role in the pathogenesis of arthritis.
In anti-CCP+RF+/? patients versus HCs a significant decrease in EV-CD14+ was observed. CD14 can be
found in two forms; a form anchored to the membrane by a glycosylphosphatidylinositol tail (mCD14) and a
soluble form (sCD14)39. It has been described that under inflammatory conditions, CD14 can be released from
monocytes membrane into the culture milieu40. Therefore, the decrease frequency of this molecule observed in
EVs from seropositive patients with RA could be based on the aforementioned phenomenon. In fact, an increase
in the sCD14 blood level in patients with RA has been reported41. Moreover, an increase in the frequency of
EV-HLA-DR+ was observed in all groups of patients with RA, together with high frequencies of platelet-derived
EVs. Although the expression of HLA-DR in platelets or platelets-derived EVs has not been observed in healthy
individuals, it has been described previously in platelets of patients with autoimmune diseases such as acute
idiopathic thrombocytopenic purpura42. A study with similar patients showed that platelets-derived EVs also
have HLA-DR expression43. Thus, the increase in EV-HLA-DR+ in patients with RA can either be related to an
increased expression of HLA-DR+ in EV-CD41a+ or could correspond to a leukocyte source. Further
experiments are required to solve these assumptions in patients with RA.
Similar to our results, Nathalie Cloutier et al.10 reported that circulating and SF EVs from patients with RA are
highly heterogeneous in size and larger than those from HCs. However, according the t-SNE diagrams, no clear
association was observed between the EV-size increase and the formation of ICs in seropositive patients with
RA. In this respect, it is important to mention that we did not exclude EV aggregation, as previously reported44.
Actually, it is striking that seropositive patients with RA have lower EVs count than seronegative ones. Although
we do not have a clear explanation for this finding, we think it is possible that the formation of ICs by these
vesicles can cause these structures to aggregate44?46; ICs activate and induce platelet aggregation47, and a considerable
frequency of platelets-derived EVs in blood of patients with RA are CD62P+ and CD40L+ (platelet activation
markers) (Villar-Vesga J, et al. Submitted manuscript). Therefore, whether ICs can promote aggregation of EVs in
a manner similar to platelets remains unknown. Additional studies are required to corroborate this hypothesis.
Previously, it was verified that EVs from the SF of RA patients form ICs through the interaction of anti-CCP with
citrullinated autoantigens10. Although we did not explore this in detail here, our results agree with this because
t-SNE analysis suggests that some EV-CPs+also were IgG+ and IgM+, mainly in seropositive patients.
In concordance with our study, other groups have demonstrated an increase in intermediate monocytes in the
circulation of patients with RA35. Interestingly, we also found this increase was present mainly in the circulation
and SF of seropositive patients and that EV-ICs were mostly captured by intermediate monocytes of RA patients
compared with cells of HCs. Other monocyte subsets have been studied in seropositive patients with RA. In a
recent study, two populations of circulating monocytes were evaluated48. M1 monocytes were defined as positive
for CD14, CD68, and CCR2, whereas M2 monocytes were defined as positive for CD14, CX3CR1, and CD163.
The authors demonstrated that patients with RA positive for ACPA had higher M1/M2 ratios. Moreover, a
positive correlation was observed between the M1/M2 ratio and the number of differentiated osteoclast detected in
vitro in samples of patients with RA. Furthermore, M1 monocytes produced higher concentrations of IL-6 upon
stimulation with lipopolysaccharide than M2 monocytes. However, because the expression of CD16 was not
studied in M1 and M2 monocytes, it is not possible at this point to compare these data with our results.
Previously, CD16+ monocytes found in the SF of RA patients have been associated with elevated levels of
proinflammatory cytokines and joint destruction17,49, and RA patients have shown a negative correlation between
intermediate monocyte frequency and disease activity scores; however, in our study we did not find this association (Figure?S2
and data not shown), instead, we found that alterations in intermediate monocytes and EVs, along with the elevated
serum concentrations of IL-6, TNF-?, and IL-1?, were closely related in seropositive patients. Actually, intermediate
monocytes exhibited more remarkable phenotypic changes in the SF of seropositive patients with RA than those
observed in the peripheral blood of the same patients. This suggests that intermediate monocytes in the joint have a
more significant involvement than their blood counterparts, which can be related to proinflammatory activation or
differentiation of these cells once they reach this location50. Additionally, EVs induced the secretion of IL1?, IL6, and
TNF-? by mononuclear phagocytes. Previously, it has been proposed that the targeting of monocytes/macrophages
should be a powerful way of inhibiting inflammation and bone erosion in RA35. Considering all of these aspects, it is
tempting to speculate that the suppression of monocyte subsets, mainly intermediate ones (for example, by avoiding
their interaction with EVs), may ameliorate the systemic inflammatory process observed in patients with seropositive
RA. Actually, the anti-TNF-? (inflixamab, adalimumab, etanercept, among others) and anti-IL-6 (tocilizumab,
sarilumab, sirukumab, among others) biological therapies, which are effective RA treatments51,52, may be partially useful
by targeting monocyte proinflammatory responses to circulating EVs. Given that, in the present study, biological
therapy was a variable of exclusion, further investigations are required to identify whether seropositive patients with
RA under anti-TNF-? and anti-IL-6 treatments have fewer EVs and monocytes alterations, possibly related with less
systemic inflammation. However, it is important to mention that the treatment received by the patients of this study
was not related to the alterations observed in EVs and monocyte subsets (Data not shown).
Finally, our results in part explain the systemic inflammatory phenomenon that is occurring in patients with
seropositive RA in which EVs and monocyte subsets appear to contribute. Seropositive patients are characterized
by an increased number of intermediate monocytes and the presence of EV-ICs+ and EV-CPs+ together with the
proinflammatory cytokines IL-1?, IL-6, and TNF-?. Therefore, the measurement of these circulating components
could be a useful approach for establishing parameters of systemic inflammation in patients with RA that are not
evaluated in any way by conventional tools, such as the DAS28.
Reagents, materials, and antibodies. RPMI-1640 medium supplemented with GlutaMAX, Dulbecco?s
phosphate-buffered saline (DPBS) and fetal bovine serum (FBS) were purchased from Gibco-BRL (Grand Island,
NY). Paraformaldehyde was acquired from Thermo Fisher Scientific, Inc. (Pittsburgh, PA). Histopaque??1077,
trypan blue, penicillin, and streptomycin were purchased from Cambrex-BioWhittaker (Walkersville, MD).
Patients and controls. Sixty patients with RA, diagnosed according to the American College of Rheumatology
criteria and European League Against Rheumatism criteria2, were recruited at the Rheumatology Service of the
Hospital Universitario San Vicente Fundaci?n (HUSVF, Medellin-Colombia); their median age was 55 years with
a range of 30 to 77 years, and 84% were women. The patients were clustered in three groups: one seronegative
[anti-CCP?RF?, (n = 6)] and two seropositive [anti-CCP+RF+/? (n = 26) and anti-CCPhiRFhi (n = 28)]; none of the
patients received biological therapy. As controls, we included 40 HCs with a median age of 46 years with a range of
25 to 77 years, and 85% were women; and 60 SLE patients with a median age of 46 with a range of 25 to 77 years, and
85% were women, only for measurement of serum cytokines19. Our study was carried out in accordance with the
Declaration of Helsinki. Research protocol and informed consent were approved by the Universidad de Antioquia?s
Medical Research Institute and HUSVF Ethics Committees. All the experiments were performed in compliance
with good clinical practice and good laboratory practice, in accordance with relevant guidelines and regulations of
Universidad de Antioquia?s Medical Research Institute. All patients and HCs provided signed informed consent.
Cytokines, autoantibodies, and CRP determination. To determine the serum cytokines IL-8, IL-6,
IL-10, TNF-?, IL-1?, and IL12p70, the BDTM Human Inflammatory CBA was used according to the
IgM and IgG RF, as well as the IgG anti-CCP, were determined in the serum of patients by commercial ELISA
(Quanta-Lite RF-IgM, Quanta-Lite RF-IgG and Quanta-Lite CCP3-IgG; Innova Diagnostics, San Diego, CA)
according to the manufacturer?s instructions. A positive result for RF was >20 IU/mL and for anti-CCP was >10
IU/mL according to the commercial house. Results between 10 and <250 IU/mL and ?250 IU/mL for these
antibodies were arbitrarily considered for the definition of the anti-CCP+RF+/? and anti-CCPhiRFhi groups, respectively.
CRP quantification was performed by immunoturbimetry using the commercial kit Reagent Dimension Flex
Quantitative Determination CRP Extended Range and the Dimension Clinical Chemistry Systems (both from
McKesson, San Francisco, CA).
Monocyte subset classification. The monocyte subsets were defined from total blood and SF by flow
cytometry as we previously reported19,53: CD14++CD16? (classical), CD14++CD16+ (intermediate), and
CD14 + CD16 ++ (non-classical) monocytes. Briefly, total blood was stained with anti-human CD45, CD14,
CD16, and HLA-DR antibodies for 20 min at room temperature in darkness; fluorescence minus one (FMO)
controls were performed for each antibody, and 100,000 cells were acquired immediately on the FACS CantoTM II
flow cytometer by using the FACS DIVA software (BD).
Monocyte subsets immunophenotyping. Mononuclear cells from blood and SF were obtained by using
a Ficoll density gradient as we previously described19. The expression of each molecule was evaluated by using
specific antibodies against CCR2, CX3CR1, CD64, CD36, CD86, CD11b, CD32, Fc?R, CD18, and HLA-DR on
monocyte subsets (basic panel CD14, CD16, and HLA-DR). FMO controls were performed for each antibody by
using the basic panel. Cells were blocked (DPBS plus 1% bovine serum albumin (BSA), 0.01% NaN3, and 10%
inactivated FBS) and stained for 30 min at 4 ?C, followed by two washes with washing buffer (DPBS plus 1% BSA
and 0.01% NaN3). Immediately, 50,000 monocytes were acquired by flow cytometry.
EVs characterization. Circulating and SF EVs were isolated and characterized as we previously described19 by
using monoclonal antibodies against human CD41a-PE, CD45-PE-Cy7, CD105-BV421, CD33-PE, CD235a-APC,
CD3-PE, CD19-V450, CD14FITC, CD16-BV450, HMGB1-APC, C1q-FITC, and HLA-DR-APC-Cy7. To measure
EV-ICs, F(ab?)2 anti-IgG-Alexa Fluor-488 and anti-IgM-Alexa Fluor-647 fragments were used. To evaluate the presence
of citrullinated peptides on EVs by flow cytometry, a specific primary antibody (rabbit polyclonal anti-human citrulline
peptides antibody), and Alexa Fluor 647-labeled secondary antibody (anti-rabbit IgG H&L-Alexa Fluor 647-secondary
antibody) were used. The EV size and count were evaluated with reference beads as we previously described19.
Some EVs samples from patients with RA and HCs were evaluated through scanning transmission electron
microscopy (STEM). For this purpose, the vesicles were fixed with 2.5% glutaraldehyde, deposited on copper-coated carbon
STEM grids; dried in the grid at 40 ?C, and contrasted with uranyl acetate and lead citrate. Finally, the samples were
dried at room temperature and evaluated in the Tecnai G2 F20 microscope (FEI company, Hillsboro, OR). Images were
recorded with an Ultra Scan 1000XP-P camera (Gatan, Inc, Pleasanton, CA).
EV-ICs formation. EVs from plasma poor-platelets of eight patients with seropositive RA were obtained and
frozen until use. For opsonization, approximately 1 ? 106 EVs were mixed and incubated with 15 ?g/mL purified
IgG (isolated from plasma of 16 RA seropositive patients by affinity chromatography) for 60min at 37 ?C. The
unbound antibodies were washed away with 1 mL of DPBS at 17,000 g for 60 min. The EV- formation was assessed
by flow cytometry (Figure?S3).
Monocyte isolation for culture with EVs and EV-ICs. Circulating monocytes were enriched by Rosette
Sep, according to the manufacturer?s instructions. The purity of the CD14+ monocytes in all cases was greater
than 90%. The binding and uptake of EVs and EV-ICs stained with 2 ?M CFSE by monocyte subsets and
phagocytes after 24 h of culture were performed at a ratio of 1:3 (cell: EVs) as reported19. To estimate the mean
fluorescence intensity (MFI) of cells that internalized EVs, the samples were acquired on a flow cytometer in the
presence or absence of 0.01% v/v trypan blue to quench the green fluorescence of surface-bound EVs.
Approximately 1.2 ? 105 monocytes were cultured as we previously described54. Briefly, the cells were adhered
in RPMI-1640-GlutaMAX with 0.5% inactivated autologous serum-depleted of EVs (iSA-f )19. Then, the cells
were incubated in RPMI-1640 with 10% iSA-f in the presence or absence of EVs and EV-ICs for 120 hours. The
supernatants were collected and frozen at ?20 ?C until cytokine assessment by CBA.
Data analysis. Comparisons among the RA groups? patients and HCs were performed by Kruskal?Wallis
test and Dunn?s post-hoc test (data as median ? interquartile range). Comparisons of more than one variable were
performed by using ANOVA-II and Bonferroni post-hoc test?(data as mean ? standard deviation). Statistical
significance was set at p ? 0.05; *p ? 0.05, **p ? 0.01, ***p ? 0.001, and ****p ? 0.0001. Correlations were made by
determining the Spearman?s rank coefficient with a 95% confidence interval. To illustrate the variations in size and
content of EVs, t-SNE analysis was applied, with perplexity set to 100, and down sampling to 50,000; t-SNE plots
were colored according to the expression of chosen channels by using ACCENSE 0.0.5-beta software55. The gating
analyses, cells/EV frequencies, and MFI were estimated by using FlowJo 10.2 software. The statistical analysis was
performed by using GraphPad Prism version 7.2 (GraphPad Soft-ware Inc., San Diego, CA). Multivariate
discriminant analysis and PCA were performed by using Statgraphics X64 Centurion XVI (Statpoint Technologies,
Warrenton, VA, USA).
The datasets generated during and/or analyzed during the current study are available from the corresponding
author on reasonable request.
C. Burbano is a recipient of a doctoral scholarship from COLCIENCIAS (call 617-2013). The authors are grateful
for grants from ?Sostenibilidad and Sistema Universitario de Investigaciones, CODI? (2013-05-42869836) from
Universidad de Antioquia, and the COLCIENCIAS project (111565740575). The authors thank Blanca Ortiz for
helping in the ELISA process, Janine Orejuela for her support in gathering the clinical data, and Natalia Ortiz and
Johana Arboleda (Transmission Electron Microscopy Facility, Universidad de Antioquia) for helping with STEM
C.B. and D.C. contributed to the acquisition, analysis, and interpretation of the data; wrote the main manuscript.
M.R. and G.V. contributed to the analysis and interpretation of the data, and drafting of the paper. C.M.-V.,
A.V.-G., and L.C. carried out the acquisition and interpretation of the clinical data. All authors reviewed and
approved the final version of the manuscript.
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36335-x.
Competing Interests: The authors declare no competing interests. Publisher?s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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1. McInnes , I. B. S. G. The pathogenesis of rheumatoid arthritis . N. Engl. J. Med . 365 , 2205 - 2219 ( 2011 ).
2. Aletaha , D. et al. Rheumatoid arthritis classifi cation criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative . Ann. Rheum. Dis . 1580 - 1588 , https://doi.org/10.1136/ard. 2010 . 138461 ( 2010 ).
3. G?mez-Puerta , J. A. et al. Differences in synovial fluid cytokine levels but not in synovial tissue cell infiltrate between anticitrullinated peptide/protein antibody-positive and -negative rheumatoid arthritis patients . Arthritis Res. Ther . 15 ( 2013 ).
4. Conigliaro , P. et al. Autoantibodies in inflammatory arthritis . Autoimmun. Rev . 15 , 673 - 683 ( 2016 ).
5. Szekanecz , Z. & Koch , A. Macrophages and their products in rheumatoid arthritis . Current opinion in rheumatology 19 ( 2007 ).
6. Hennigan , S. & Kavanaugh , A. Interleukin-6 inhibitors in the treatment of rheumatoid arthritis . Ther. Clin. Risk Manag . 4 , 767 - 775 ( 2008 ).
7. Toussirot , E. & Wendling , D. The use of TNF-alpha blocking agents in rheumatoid arthritis: an update . Expert Opin. Pharmacother. 8 , 2089 - 2107 ( 2007 ).
8. Edalat , F. NIH Public Access . 40 , 1301 - 1315 ( 2012 ).
9. Boilard , E. et al. Platelets amplify inflammation in arthritis via collagen-dependent microparticle production . Science 327 , 580 - 3 ( 2010 ).
10. Cloutier , N. et al. The exposure of autoantigens by microparticles underlies the formation of potent inflammatory components: The microparticle-associated immune complexes . EMBO Mol. Med . 5 , 235 - 249 ( 2013 ).
11. Malda , J. , Boere , J., van de Lest, C. H. A., van Weeren, P. R. & Wauben , M. H. M. Extracellular vesicles - new tool for joint repair and regeneration . Nature reviews. Rheumatology 12 , 243 - 249 ( 2016 ).
12. Burbano , C. , Rojas , M. , V?squez , G. & Casta?o , D. Microparticles That Form Immune Complexes as Modulatory Structures in Autoimmune Responses . Mediators Inflamm . 2015 ( 2015 ).
13. Stuhlm?ller , B. et al. Identification of known and novel genes in activated monocytes from patients with rheumatoid arthritis . Arthritis Rheum . 43 , 775 - 790 ( 2000 ).
14. Liote , F. , Boval-Boizard , B. , Weill , D. , Kuntz , D. & Wautier , J. L. Blood monocyte activation in rheumatoid arthritis: increased monocyte adhesiveness, integrin expression , and cytokine release. Clin. Exp. Immunol . 106 , 13 - 19 ( 1996 ).
15. Torsteinsdottir , I. , Arvidson , N. G. , Hallgren , R. & Hakansson , L. Monocyte activation in rheumatoid arthritis (RA): increased integrin, Fc gamma and complement receptor expression and the effect of glucocorticoids . Clin. Exp. Immunol . 115 , 554 - 560 ( 1999 ).
16. Burmester , G. R. et al. The tissue architecture of synovial membranes in inflammatory and non-inflammatory joint diseases. I. The localization of the major synovial cell populations as detected by monoclonal reagents directed towards Ia and monocytemacrophage antigens . Rheumatol. Int. 3 , 173 - 181 ( 1983 ).
17. Kinne , R. W. , Stuhlmuller , B. & Burmester , G.-R. Cells of the synovium in rheumatoid arthritis . Macrophages. Arthritis Res. Ther . 9 , 224 ( 2007 ).
18. Nielsen , C. T. et al. Increased IgG on cell-derived plasma microparticles in systemic lupus erythematosus is associated with autoantibodies and complement activation . Arthritis Rheum . 64 , 1227 - 1236 ( 2012 ).
19. Burbano , C. et al. Potential involvement of platelet-derived microparticles and microparticles forming immune complexes during monocyte activation in patients with systemic lupus erythematosus . Front. Immunol . 9 , ( 2018 ).
20. Rossol , M. , Kraus , S. , Pierer , M. , Baerwald , C. & Wagner , U. The CD14 brightCD16+ monocyte subset is expanded in rheumatoid arthritis and promotes expansion of the Th17 cell population . Arthritis Rheum . 64 , 671 - 677 ( 2012 ).
21. Withrow , J. et al. Extracellular vesicles in the pathogenesis of rheumatoid arthritis and osteoarthritis . Arthritis Res. Ther . 18 , 286 ( 2016 ).
22. Pisetsky , D. S. , Ullal , A. J. , Gauley , J. & Ning , T. C. Microparticles as mediators and biomarkers of rheumatic disease . Rheumatol . (United Kingdom) 51 , 1737 - 1746 ( 2012 ).
23. Shi , C. & Pamer , E. G. Monocyte recruitment during infection and inflammation . Nat. Rev. Immunol . 11 , 762 - 774 ( 2011 ).
24. Scrivo , R. et al. Distribution of interleukin-10 family cytokines in serum and synovial fluid of patients with inflammatory arthritis reveals different contribution to systemic and joint inflammation . Clin. Exp. Immunol . 179 , 300 - 308 ( 2015 ).
25. Sattar , N. , McCarey , D. W. , Capell , H. & McInnes , I. B. Explaining How 'High-Grade' Systemic Inflammation Accelerates Vascular Risk in Rheumatoid Arthritis . Circulation 108 , 2957 - 2963 ( 2003 ).
26. Ohmura , K. et al. Anti-citrullinated peptide antibody-negative RA is a genetically distinct subset: a definitive study using only boneerosive ACPA-negative rheumatoid arthritis . Rheumatology (Oxford) . 49 , 2298 - 2304 ( 2010 ).
27. van der Woude , D. et al. The ACPA isotype profile reflects long-term radiographic progression in rheumatoid arthritis . Ann. Rheum. Dis . 69 , 1110 - 1116 ( 2010 ).
28. Gonz?lez-?lvaro , I. et al. Interleukin 15 levels in serum may predict a severe disease course in patients with early arthritis . PLoS One 6 , 1 - 9 ( 2011 ).
29. MU , R. et al. Elevated Serum Interleukin 33 Is Associated with Autoantibody Production in Patients with Rheumatoid Arthritis . J. Rheumatol. 37 , 2006 - 2013 ( 2010 ).
30. Gorlino , C. V. et al. Association between levels of synovial anti-citrullinated peptide antibodies and neutrophil response in patients with rheumatoid arthritis . Eur. J. Immunol . 1563 - 1572 , https://doi.org/10.1002/eji.201847477 ( 2018 ).
31. Krishnamurthy , A. et al. Identification of a novel chemokine-dependent molecular mechanism underlying Rheumatoid arthritisassociated autoantibody-mediated bone loss . Ann. Rheum. Dis . 75 , 721 - 729 ( 2016 ).
32. Wigerblad , G. et al. Autoantibodies to citrullinated proteins induce joint pain independent of inflammation via a chemokinedependent mechanism . Ann. Rheum. Dis. 75 , 730 - 738 ( 2016 ).
33. Catrina , A. I. , Svensson , C. I. , Malmstrom , V. , Schett , G. & Klareskog , L. Mechanisms leading from systemic autoimmunity to jointspecific disease in rheumatoid arthritis . Nat. Rev. Rheumatol . 13 , 79 - 86 ( 2017 ).
34. White , F. A. , Jung , H. & Miller , R. J. Chemokines and the pathophysiology of neuropathic pain . Proc. Natl. Acad. Sci . 104 , 20151 - 20158 ( 2007 ).
35. Davignon , J.-L . et al. Targeting monocytes/macrophages in the treatment of rheumatoid arthritis . Rheumatology (Oxford) . 52 , 590 - 598 ( 2013 ).
36. McInnes , I. B. Rheumatoid arthritis. From bench to bedside . Rheum. Dis. Clin. North Am . 27 , 373 - 387 ( 2001 ).
37. Baron , M. , Boulanger , C. M. , Staels , B. & Tailleux , A. Cell-derived microparticles in atherosclerosis: biomarkers and targets for pharmacological modulation? J . Cell . Mol. Med . 16 , 1365 - 1376 ( 2012 ).
38. del Rincon , I. D. , Williams , K. , Stern , M. P. , Freeman , G. L. & Escalante , A. High incidence of cardiovascular events in a rheumatoid arthritis cohort not explained by traditional cardiac risk factors . Arthritis Rheum . 44 , 2737 - 2745 ( 2001 ).
39. Kirkland , T. N. & Viriyakosol , S. Structure-function analysis of soluble and membrane-bound CD14 . Prog. Clin. Biol. Res . 397 , 79 - 87 ( 1998 ).
40. Bazil , V. & Strominger , J. L. Shedding as a mechanism of down-modulation of CD14 on stimulated human monocytes . J. Immunol . 147 , 1567 - 1574 ( 1991 ).
41. Mikuls , T. R. et al. Soluble CD14 and CD14 polymorphisms in rheumatoid arthritis . J. Rheumatol . 38 , 2509 - 2516 ( 2011 ).
42. Boshkov , L. K. , Kelton , J. G. & Halloran , P. F. HLA-DR expression by platelets in acute idiopathic thrombocytopenic purpura . Br. J. Haematol . 81 , 552 - 557 ( 1992 ).
43. Semple , J. W. et al. Differences in serum cytokine levels in acute and chronic autoimmune thrombocytopenic purpura: relationship to platelet phenotype and antiplatelet T-cell reactivity . Blood 87 , 4245 - 4254 ( 1996 ).
44. Gyorgy , B. et al. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle) signatures in joint diseases . PLoS One 7 , e49726 ( 2012 ).
45. Mustonen , A.-M . et al. First in vivo detection and characterization of hyaluronan-coated extracellular vesicles in human synovial fluid . J. Orthop. Res . 34 , 1960 - 1968 ( 2016 ).
46. Khlebtsov , B. N. , Burygin , G. L. , Matora , L. Y. , Shchyogolev , S. Y. & Khlebtsov , N. G. A method for studying insoluble immune complexes . Biochim. Biophys. Acta 1670 , 199 - 207 ( 2004 ).
47. Schattner , M. et al. Activation of human platelets by immune complexes prepared with cationized human IgG . Blood 82 , 3045 - 3051 ( 1993 ).
48. Iwamoto , N. M1 and M2 Monocytes in rheumatoid arthritis: a contribution of imbalance of M1/M2 Monocytes to Osteoclastogenesis . 8 , 1 - 10 ( 2018 ).
49. Iguchi , T. , Kurosaka , M. & Ziff , M. Electron microscopic study of HLA-DR and monocyte/macrophage staining cells in the rheumatoid synovial membrane . Arthritis Rheum . 29 , 600 - 613 ( 1986 ).
50. Wynn , T. A. , Chawla , A. & Pollard , J. W. Macrophage biology in development, homeostasis and disease . Nature 496 , 445 - 455 ( 2013 ).
51. Md Yusof , M. Y. & Emery , P. Targeting interleukin-6 in rheumatoid arthritis . Drugs 73 , 341 - 356 ( 2013 ).
52. Neovius , M. et al. Drug survival on TNF inhibitors in patients with rheumatoid arthritis comparison of adalimumab, etanercept and infliximab . Ann. Rheum. Dis. 74 , 354 - 360 ( 2015 ).
53. Burbano , C. , Vasquez , G. & Rojas , M. Modulatory effects of CD14+CD16++ monocytes on CD14++CD16? monocytes: a possible explanation of monocyte alterations in systemic lupus erythematosus . Arthritis Rheumatol . (Hoboken, N.J.) 66 , 3371 - 3381 ( 2014 ).
54. Casta?o , D. , Garc?a , L. F. & Rojas , M. Increased frequency and cell death of CD16+monocytes with Mycobacterium tuberculosis infection . Tuberculosis 91 , 348 - 360 ( 2011 ).
55. Shekhar , K. , Brodin , P. , Davis , M. M. & Chakraborty , A. K. Automatic Classification of Cellular Expression by Nonlinear Stochastic Embedding (ACCENSE) . Proc. Natl. Acad. Sci . 111 , 202 - 207 ( 2014 ).