Platelets: a potential target for rheumatoid arthritis treatment?

KEYWORDS:

• Rheumatoid arthritis
• platelets
• T-cells
• microparticles
• Th1
• Th17
• Treg

1. Introduction

Platelets (PLTs) represent the second most abundant component of peripheral blood, after erythrocytes. Despite being essential contributors to blood clotting, their non-hemostatic role is very well recognized. They arise in the bone marrow from megakaryocytes and have a short lifespan due to their lack of the nucleus. Although they were originally considered mere portions of cells, there is now clear evidence of a major metabolic activity. PLTs indeed contain cellular organelles such as mitochondria (thus mitochondrial DNA), lysosomes and portions of the Golgi apparatus. They also store a large amount of RNA which allows them to respond to external stimuli with protein synthesis and to exert a control on translation via microRNAs. Their main functions are mediated by the release of cytokines, chemokines, and growth factors stored in α-granules (such as Von Willebrand factor – vWF, P-selectin – P-sel, platelet factor 4 – PF4, transforming growth factor β – TGF-β, platelet-derived growth factor – PDGF) and dense granules (namely ADP, serotonin, histamine, etc.). PLTs can also synthesize lipid mediators from membrane arachidonic acid. The release of granules is induced by platelet activation by external stimuli. The most typical example of such stimuli is collagen, which becomes exposed in case of tissue injury, activating a cascade of events eventually leading to hemostasis [1].

Nonetheless, PLTs also express class I major histocompatibility complex, toll-like receptors, receptors for the Fc fragment of immunoglobulins, glycoprotein complex receptors. Thus they can bind damage-associated molecular patterns, pathogen-associated molecular patterns, immune complexes (ICs) and molecules involved in hemostasis, such as vWF and fibrinogen [2].

2. Platelet-derived microparticles

A very interesting aspect of PLTs biology is represented by PLT-derived microparticles (MPs). MPs are small vesicles (0.1–1 μm) actively released by cells and containing structures that belong to the parent cells, including cytoplasmic organelles and membrane receptors, thus partially retaining their functions. An intuitive consequence of this phenomenon is a larger overall cell membrane surface and, consequently, a wider area available for PLT-cell interactions, and improved migration ability of MPs compared to PLTs. However, besides the different behaviors due to the different size, it is not well-known whether the role of MPs is distinct from that of PLTs [3]. Although MPs have been shown to be abundant in the synovial fluid (SF) and synovium of RA patients [4], still too little is known on their role and this aspect is worth careful and detailed exploration.

3. Definition and control of platelet activation status

The large majority of circulating PLTs are never recruited during their short lifespan and remain quiescent until they become senescent and are eliminated by the reticuloendothelial system. When activated, PLTs release MPs and granules’ content and synthesize molecules such as thromboxane (TX)A2 from membrane phospholipids and interleukin (IL)-1 from constitutive mRNA derived from megakaryocytes. Activated PLTs are conventionally identified as PLTs expressing high levels of membrane P-sel, CD40 ligand (CD40L), which is eventually cleaved and released in its soluble form sCD40L, and producing TXA2, although numerous other events take place. It is now widely accepted that the response of PLTs is stimulus-specific, that is they respond differently according to the stimulus received. Unfortunately, very little is known on the mechanism underlying such precise control of PLT functions; thus the use of a dichotomous classification of PLT status as inactive or activated, as commonly defined in experimental settings, is largely an oversimplification which likely leads to a hazy interpretation of reality. It is highly likely that the concept of activation will be revised in the future as more evidence accumulates that probably more ‘types of activations’ exist.

4. Interactions of PLTs with leukocytes

Extensive evidence shows that PLTs can bind to leukocytes, including monocytes, neutrophils, and T cells [5,6]. Even more, interestingly, the number of circulating leukocytes-PLTs aggregates increases following PLTs activation. The number of circulating activated PLTs is increased in patients with autoimmune inflammatory diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis. Additionally, some receptors expressed by PLTs do not appear to have a role in the complex system of hemostasis and thrombosis [7,8].

All considered, it is intuitive to suppose that PLTs are closely involved in immune regulation through humoral and cell-mediated mechanisms. Upon binding, PLTs influence leukocytes and vice versa. Neutrophils can bind PLTs through various receptors, including CD162 (P-sel ligand), CD40, and CCL5 (PF4 ligand) and are eventually stimulated to produce neutrophil extracellular traps (NETs) which, in turn, further activate PLTs. Neutrophils are also considered among the most important carriers that allow PLTs extravasation toward target tissues [9].

We have recently demonstrated that PLTs and MPs present in SF of osteoarthritis (OA) patients are also capable of binding fibroblast-like synoviocytes (FLS) via the interaction of P-sel with CD44 and stimulate them to produce matrix metalloproteinase (MMP)2, which is responsible for cartilage degradation and joint damage. Interestingly, intra-articular injections with hyaluronic acid (HA) were able to reduce the amount of MMP2 in SF by hampering the interaction of PLTs with FLS [10]. Current ongoing experiments seem to confirm a similar phenomenon in RA. Although such evidence perfectly fits with the positive outcomes of intra-articular HA use in OA, it does not explain why platelet-rich plasma (PRP) can be used in the same group of patients to improve symptoms of OA [11]. Moreover, intra-articular PRP injections were able to reduce inflammation in a pig model of inflammatory arthritis [12]. Many variables could account for such divergent observations, such as a potential difference in terms of PLT activation status.

One of the most interesting aspects to investigate about PLTs in the field of autoimmune diseases is their interaction with lymphocytes, which is mediated by several receptors, such as P-sel, CD40 and GPIIb/IIIa [5]. Available published data describe some very interesting phenomena worth discussing. Some papers show that when CD3/CD28-stimulated CD4⁺ T-cells are cultured with PLTs at a PLTs:T-cell ratio of 250:1, their proliferation is hampered, but differentiation towards Th1, Th17, and Treg phenotypes is markedly increased. The effect of PLTs is stronger when they are allowed to directly interact with T-cells, but it is still significant when contact is prevented, thereby demonstrating that soluble mediators such as TGF-β and PF4 – and perhaps MPs – are involved as well [13]. Nonetheless, when co-cultures are extended beyond day 3, the percentage of Th1 and Th17 cells significantly drops, while the effect on Tregs is maintained. Moreover, as proliferation rate is analyzed in the three T-cell subtypes, unlike Th1 and Th17 cells, that of Tregs is increased [14]. Other studies apparently show partially different results. When peripheral blood mononuclear cells (PBMCs) are cultured with PLTs, a reduction of IFNγ and TNFα in supernatants, compared to PBMCs alone, is observed and such a reduction is stronger when a higher PLTs:PBMCs ratio is used in culture (up to 100:1). When the same experiment is performed using RA synovial fluid derived mononuclear cells, in addition to TNFα and IFNγ, IL-17 is reduced in culture supernatants as well, while IL-10 production increases [15].

Interpretation of these data is a very complex exercise which will likely lead to inconclusive results. As we previously described, PLTs are very sensitive to external stimuli and therefore difficult to use in vitro. Thus, divergent results could be influenced not only by the characteristics of the donors but also by the ex vivo and in vitro processing of the samples. The experimental conditions are of substantial importance in order to obtain consistent results. Numerous methodological variables need to be considered, such as venipuncture technique, blood tubes used (Li-heparin, Na-citrate), any additional substances used to prevent PLTs activation (prostacyclin, COX-inhibitors), PLTs extraction method, culture medium used (RPMI, DMEM), time of incubation, co-culture cell ratio, type of cells used for co-culture (CD4⁺ T-cells, PBMCs) and origin of PLTs (PB or SF). Even minor differences in any of these steps may generate non comparable results. Additionally, it is still unknown what the best method is in order to accurately reproduce the in vivo phenomena. Thus, even with an excellent internal validity, any in vitro observation could be very different from reality. All these aspects should very carefully be taken into account for the design and analysis of future experiments.

Nonetheless, there are a few deductions that could be made with a fair degree of certainty. Firstly, PLTs most likely have a dual role when involved in the regulation of immunity, i.e. they can be both pro-inflammatory and anti-inflammatory and such difference is probably dependent on the environment (e.g. number of platelets (PLTs), type of cells interacting, culture time), although specific triggers have not yet been identified. Secondly, the regulation of thrombotic and immune functions of PLTs are likely distinct mechanisms, though with a certain degree of interrelation. However, even considering the extreme sensitivity of platelets (PLTs) to experimental conditions, their effects in terms of immune regulation are by any means very complex.

5. PLTs as therapeutic targets

Since numerous medications targeting PLTs are available for the treatment and prevention of thromboembolism, the first question that arises is whether any of these drugs can have an effect in regulating autoimmunity. Unfortunately, scientific evidence is very limited, mostly because these mechanisms have not been extensively investigated. Some drugs indeed have a role in the prevention of cardiovascular events in patients with autoimmune diseases, like aspirin in antiphospholipid syndrome. Another group of popular antiplatelet drugs is ADP receptor P2Y₁₂ inhibitors, such as clopidogrel, which, by blocking the release of α-granules, was shown to reduce levels of P-sel, CD40L, C-reactive protein, and TNFα, but was only studied on patients with cardiovascular disease. Nonetheless, clopidogrel has been associated with episodes of acute arthritis, whose pathogenesis is probably independent from P2Y₁₂ activity, thus the effect of the drug is likely pleiotropic [16]. Two trials are currently ongoing evaluating the effect of P2Y₁₂ inhibitors in RA (ticagrelor, NCT02874092) and SLE (clopidogrel, NCT02320357), but results are not yet available.

Another interesting target is CD40L, whose blockade (ruplizumab and dapirolizumab pegol) has demonstrated benefits in lupus nephritis, although the trial with ruplizumab was terminated prematurely due to an unacceptable increase of cardiovascular events [17,18]. It is interesting to note that, despite data are too preliminary to draw conclusions, an increase in thromboembolic events following blockade of CD40L would further confirm how the mechanisms controlling hemostasis and immunity in PLTs are distinct but somehow not independent.

Some other well-known drugs, already commonly used to treat various conditions, are known to have an effect on PLTs, such as hydroxychloroquine [7], statins [19] and selective serotonin reuptake inhibitors [20], though they have never been formally evaluated in the context of PLTs function in inflammatory diseases.

6. Conclusions

PLTs represent a very interesting player in autoimmune and inflammatory diseases and deserve careful scientific consideration. Unfortunately, their great complexity and pleiotropy represents a double-edge sword. On the one hand, it makes them a very attractive actor in autoimmunity from a pathogenic and therapeutic point of view, on the other hand, it makes obtaining high quality experimental evidence a great challenge. We already have a deep knowledge of their function in hemostasis and we have been able to detect a wide range of targets to modulate their function. Some of the molecules and targets already known may in fact be useful to treat autoimmunity, but need to be approached from another perspective and new mechanisms should be further investigated to expand our knowledge and improve the quality of treatment of autoimmune diseases.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This article was not funded.