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Impaired microRNA processing in RA neutrophils
various single nucleotide polymorphism have been stud- ied in miRNA in RA.15
Our study shows a global downregulation of the miRNA expression in RA neutrophils, more marked in SF neutrophils, suggesting that it might contribute to the abnormal activated profile of these cells in the synovium.
In this sense, global downregulation of miRNA has been shown in human alveolar macrophages induced by cigarette smoking, responsible for the changes in gene expression associated with the disease.16 In autoimmune disorders, we recently described a global downregulation of the miRNA levels in neutrophils from patients with systemic lupus erythematosus and antiphospholipid syn- drome, which may indicate that chronic inflammation and/or autoimmunity is associated with a reduction of miRNA in neutrophils.17 In the present study, we demon- strate that either ACPA or inflammatory mediators, espe- cially TNF-a can modulate the miRNA expression pro- file, through a reduction of several proteins involved in its processing, which might be translated into an increase of genes that might be involved in inflammation, cell sur- vival and migration.
Up to date, no study has reported the effect of ACPA on the expression of miRNA. In our hands, the reduced levels of miRNA and DICER in RA neutrophils correlated with elevated levels of ACPA. Accordingly, our in vitro studies demonstrated a direct involvement of these autoantibodies in the deregulation of various miRNA - and their specific protein targets- globally related to the pathogenesis of RA. We further demonstrated that the global downregulation of the miRNA expression in RA neutrophils was associated, at least partially, with the reduced levels of DICER. A recent study suggested the role of DICER in neutrophils differentiation, where the DICER inhibition attenuated the activation of autophagy, a process that is needed for proper neutrophil differentia- tion.12 DICER plays a crucial role in miRNA biogenesis. Thus, it has been suggested that mRNA and protein levels of DICER must be strictly controlled since small changes can initiate various pathological processes.18 Here, we prove a novel role for DICER in neutrophils, showing that a small reduction of protein levels can induce a proinflam- matory profile in neutrophils by downregulating several miRNA and, hence, a number of putative targeted cytokines and chemokines.
Currently, little is known about the miRNA regulating neutrophil function. Several miRNA have recently been involved in the neutrophil development and function and in various pathological states, including miRNA-155, miRNA-34a, miRNA-223, miRNA-142, miRNA-452 and miRNA-466L.19 Overexpression of miRNA-155 and miRNA-34a in neutrophils from patients with myelodys- plastic syndrome has been shown to contribute to an alteration of the migration.20 In addition, decreased levels of both, the miRNA-145 and the miRNA-143 have been shown in acute myelogenous leukemia, which are responsible for the blockade of the differentiation process of the neutrophils.21
Alongside with previous evidence, here we show that miRNA-223 is one of the most abundant miRNA on neu- trophils.22 It has recently been demonstrated that the miRNA-223 is an important regulator blocking the infil- tration of neutrophils in alcoholic hepatic disease.22 Supporting a role for this miRNA in the infiltration capac- ity of the neutrophils, in the present work we demon-
strated how the overexpression of the miRNA-223 in neutrophils of RA patients reduced specifically the expression of IL-1 and IL-8, molecules involved in inflammation and migration.
The role of the miRNA-126 in the vascular integrity has been also evidenced.23 We observed reduced levels of the miRNA-126 in RA neutrophils, while its induced overex- pression in RA neutrophils significantly reduced the levels of VEGF, pointing to the role of this miRNA in neutrophil adhesion and migration.
Multiple functions have been attributed to miRNA- 148a in several diseases and low levels of miRNA-148 have been shown to be related to less survival time and increased recurrence risk in bladder cancer.24 In addition, miRNA-148 has been related to innate and adaptive immune responses.25 Our data is in agreement with these studies, since we found reduced levels of the miRNA-148 in RA neutrophils, associated to increased levels of TNF-
, a key inflammatory protein driving the RA disease. Other miRNA found decreased in RA neutrophils, such as miRNA-21, Let-7 and miRNA-30, have previously been reported to be altered in different types of tumors, thus playing a relevant role in tumorigenesis, invasion and metastasis of cancer cells.26-29 In addition, Let-7 and miRNA-17 regulate the T-cell response.27,30 Finally, a recent study demonstrated that the levels of all the mem- bers of the miRNA29 family were decreased in PB mononuclear cells and CB34+ cells of the bone marrow of acute myelogenous leukemia patients. The normalization of their levels partially inhibited the abnormal prolifera- tion of the blasts, blocked the myeloid differentiation and
repressed the apoptosis.31
TNF-a and IL-6 are key inflammatory effectors in RA,
whose levels are elevated in RA serum and further increased in RA SF. We found a marked effect of TNF-a on neutrophils, lowering the expression of genes related to miRNA processing (including DICER and AGO-1) and downregulating the eight miRNA selected. By contrast, IL-6 had less effect but was still able to reduce the levels of miRNA-126, let-7b and miRNA-17 alongside with the expression of AGO-1 and AGO-2. Treatment of active RA neutrophils with IFX or TCZ reduced the inflamma- tory profile, by downregulating the gene expression of VEGFA, TNF-a, IL-1β, IL8, IL6R and STAT3. However, only IFX was able to restore the global levels of selected miRNA, alongside with genes involved in their process- ing in RA neutrophils, an effect that might be expected after the efficciency of TNF-a observed to reduce the miRNA levels, thus suggesting that IFX might specifically minimize the abnormal profile of the RA neutrophils through the inhibition of TNF-a, which directly acts by reducing the expression of miRNA. In agreement with these results, we recently demonstrated that in vivo treat- ment with anti-TNF-a drugs for six months regulated the levels of several miRNA in the plasma of RA patients. Moreover, miRNA-23 and miRNA-223 were identified as potential biomarkers of therapy effectiveness.32
Altogether, our study shows that neutrophils from RA patients have a defect in the miRNA biogenesis machin- ery, are more marked in SF neutrophils, and are induced by ACPA and inflammatory mediators. This defect might be directly associated with the abnormal neutrophil acti- vation, thus increasing their proinflammatory profile, observable by the higher expression of a number of chemokines and cytokines.
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