Platelets Play Differential Role During the Initiation and Progression of Autoimmune NeuroinflammationNovelty and Significance
Rationale: Platelets are known to participate in vascular pathologies; however, their role in neuroinflammatory diseases, such as multiple sclerosis (MS), is unknown. Autoimmune CD4 T cells have been the main focus of studies of MS, although the factors that regulate T-cell differentiation toward pathogenic T helper-1/T helper-17 phenotypes are not completely understood.
Objective: We investigated the role of platelets in the modulation of CD4 T-cell functions in patients with MS and in mice with experimental autoimmune encephalitis, an animal model for MS.
Methods and Results: We found that early in MS and experimental autoimmune encephalitis, platelets degranulated and produced soluble factors serotonin (5-hydroxytryptamine), platelet factor 4, and platelet-activating factor, which specifically stimulated differentiation of T cells toward pathogenic T helper-1, T helper-17, and interferon-γ/interleukin-17–producing CD4 T cells. At the later stages of MS and experimental autoimmune encephalitis, platelets became exhausted in their ability to produce proinflammatory factors and stimulate CD4 T cells but substantially increased their ability to form aggregates with CD4 T cells. Formation of platelet–CD4 T-cell aggregates involved the interaction of CD62P on activated platelets with adhesion molecule CD166 on activated CD4 T cells, contributing to downmodulation of CD4 T-cell activation, proliferation, and production of interferon-γ. Blocking of formation of platelet–CD4 T-cell aggregates during progression of experimental autoimmune encephalitis substantially enhanced proliferation of CD4 T cells in the central nervous system and the periphery leading to exacerbation of the disease.
Conclusion: Our study indicates differential roles for platelets in the regulation of functions of pathogenic CD4 T cells during initiation and progression of central nervous system autoimmune inflammation.
Platelets play an important role in thrombosis and homeostasis, but their role in neuroinflammatory and neurodegenerative diseases is yet to be determined.1–3 Recently, it has been demonstrated that platelets contributed to inflammation in several conditions, including infection, rheumatoid arthritis, and arthrosclerosis.4–7 Because activated platelets secrete chemokines, cytokines, and other proinflammatory mediators, they have the capacity to initiate and regulate inflammation at the site of injury.8 Platelet-derived factors have been shown to play a significant role in both inflammation and tissue repair, suggesting an important role of platelets in the initiation, progression, and resolution of inflammation.9 One of the proposed mechanisms of regulation of inflammation by platelets is their direct interaction with leukocytes.1
Platelet–leukocyte interactions have been a focus of investigations aimed at understanding the pathology of inflammatory diseases.4,10 Platelet–leukocyte aggregates were shown to occur in pathological processes associated with vascular abnormalities, thrombosis, and inflammation.10,11 It was found that platelets interact with leukocytes, and these interactions could modulate leukocyte function.10,12,13 However, little is known about whether platelets affect the function of autoimmune CD4 T cells during neurological diseases, such as multiple sclerosis (MS).
MS is an inflammatory disease of the central nervous system (CNS), which affects young adults and can lead to significant disability. MS and its animal model, experimental autoimmune encephalitis (EAE), involve pathogenic T helper (Th)-1, Th17, and recently discovered interferon-γ (IFN-γ)/interleukin (IL)-17 double-positive CD4 T cells that recognize CNS self-antigens within myelin sheath, such as myelin oligodendrocyte glycoprotein (MOG).14–16 MS usually begins as a relapsing remitting form, which is characterized by unpredictable relapses followed by periods of remission. Within a 10- to 15-year period, many patients with relapsing remitting form enter the stage of secondary progressive MS, in which there is progressive neurological decline without remission.17 Current Food Drug Administration–approved drugs for MS are primarily for relapsing forms of the disease. These drugs are only partially effective, and many have side effects.18 There are no treatments for MS that targets platelets19; however, we have recently showed that Food Drug Administration–approved drug Copaxone affected platelet activation.20 Thus, the study of interactions of platelets with CD4 T cells has the potential to identify new diagnostic and therapeutic targets for MS.
It was demonstrated several decades ago that platelets may become activated during MS.21 Platelet abnormalities, such as thrombocytopenia, are 25× more common among patients with MS when compared with the general population.22 A more recent study reported that in patients with MS, there are increased numbers of platelet–platelet aggregates, platelet-derived microparticles, and increased expression of the activation marker CD62P on platelets.23 Taken together with reports that platelet-specific αIIbβ3 and platelets themselves are found in CNS inflammatory lesions in MS,24,25 these data suggest that platelets may be actively involved in MS pathogenesis both in the CNS and the periphery. Our studies, along with the study by Langer et al,25,26 demonstrated that platelets accumulate in CNS inflammatory lesions of mice with EAE and that depletion of platelets ameliorated CNS autoimmune inflammation in rodents at early stages of the disease. Our previous study also identified an important link between platelet activation and degranulation (ie, secretion of the contents of a platelet’s granules, such as serotonin) and damaged neuronal tissue.26 We found that platelets become activated by sialated brain-specific glycolipids integrated into neuronal and astroglial lipid rafts (LRs; brain LRs),26 which become accessible to platelets because of alterations of blood–brain barrier permeability during EAE and MS.27,28
In this study, we investigated the functional ability of the platelets from patients with MS versus healthy control (HC) subjects to degranulate and secrete serotonin (5-hydroxytryptamine [5HT]) in response to brain LRs, as well as the interaction of platelets with CD4 T cells. We found that early in MS or EAE, platelets exhibited distinct proinflammatory properties by secreting soluble factors 5HT, platelet-activating factor (PAF), and PF4 that stimulate proliferation and differentiation of pathogenic Th1, Th17, and IFN-γ/IL-17 double-positive CD4 T cells. However, at more advanced stages of the disease, platelets decreased 5HT content in the granules but increased the capacity to aggregate with CD4 T cells via interactions of CD62P on platelets with adhesion molecules activated leukocyte cell adhesion molecule/CD166 on CD4 T cells resulting in downmodulation of T-cell activation and suppression of CNS autoimmune inflammation.
Peripheral blood samples were obtained from HC subjects and patients with MS (Methods section in detail is available in the Online Data Supplement).
Platelet and Mononuclear Cell Isolation and Analysis
For isolation and analysis of human platelets, peripheral blood samples were drawn using collecting tubes with EDTA. Whole blood and platelet-rich plasma were analyzed by multicolor flow cytometry. For isolation of mononuclear cells from human subjects or mice, the Ficoll or Percoll density gradients were used according to the standard protocols (Methods section in the Online Data Supplement).10,26,29,30
Human CD4 T cells were isolated from peripheral blood mononuclear cells by negative selection using a magnetic separation and stimulated for 6 days with anti-CD3/CD28 antibodies. Cytokine production in culture supernatants was measured by ELISA as described.31 Activation of human or mouse platelets with thrombin or ADP was performed as described (Methods in the Online Data Supplement).26
C57BL/6 mice were purchased from Jackson Laboratories. B6.MOG T-cell receptor transgenic mice were maintained in our colony. For CD4 T-cell recall response, MOG T-cell receptor transgenic mice were immunized with MOG/complete Freund adjuvant.26 EAE was induced C57BL/6 mice as described earlier (Methods in the Online Data Supplement).26
Flow Cytometry Analysis of Expression of Intracellular Molecules
For intracellular detection of IL-17, IFN-γ, IL-4, or FoxP3, mouse or human CD4 T cells were activated with phorbol myristate acetate and ionomycin in the presence of GolgiStop for 4 hours. Cells were immediately stained for surface markers, fixed/permeabilized, and stained for intracellular antigens with proper antibodies directly conjugated with fluorophores. Fluorescence-activated cell sorter analysis was performed as described previously (Methods in the Online Data Supplement).32–34
Proliferation assays were performed as described in our previous studies (Methods in the Online Data Supplement).32
Unpaired Student t test and Mann–Whitney U test were used to determine the significance. P values of <0.05 were considered significant (Methods section in detail is available in the Online Data Supplement).
Platelets From Patients With MS Form Aggregates With CD4 T Cells
In our previous study, we found that platelets promote neuroinflammation and communicate with immune cells.26 On the basis of this, we hypothesized that platelets could directly interact with CD4 T cells and form platelet–CD4 T-cell aggregates. We tested this hypothesis and found that the average percentage of CD4 T cells that were aggregated with platelets was significantly increased (2.4-fold) in the peripheral blood of patients with MS when compared with HC subjects (Figure 1A). These data demonstrate an active interaction of platelets with CD4 T cells during MS.
Platelets Degranulate During MS But Fail to Secrete 5HT in Response to Stimulation by Brain LRs
In addition to increased adhesiveness to leukocytes, activated platelets have abilities to degranulate and secrete a number of proinflammatory substances, such as 5HT,26 which are stored in their granules. It was reported that degranulating platelets produce microparticles during MS23 and EAE26 and become positive for annexin V, which indicates ongoing processes of platelet activation and vesiculation.35 We found that platelets from patients with MS significantly increased (≈2-fold increase for the mean level) the annexin-V staining (Figure 1B). In addition, we confirmed a reported increase23 in the proportion of platelet-derived microparticles in the peripheral blood of patients with MS (Online Figure I), indicating platelet degranulation during MS. We also found 2.8-fold decrease in the 5HT level in the platelets of patients with MS when compared with that of HC (Figure 1C), indicating possible release of 5HT from platelet’s granules. Further analysis showed a 2-fold higher background level of 5HT in platelet-free plasma of patients with MS versus HC (Figure 1D, left, background serotonin release). Platelets are the main and the only source of serotonin in the peripheral blood, whereas 5HT originates from enterochromaffin cells in the gut, and then it is taken up by platelets.36 Therefore, 2.8-fold decrease in the 5HT content in MS platelets, a 2-fold increase in the level of 5HT in platelet-free plasma, a 2-fold increase in annexin-V expression on circulating platelets, and an increase in the number of platelet-derived microparticles in the peripheral blood of patients with MS indicate an active process of platelet degranulation during MS.
In our previous study, we showed that brain LRs play a role in the activation of mouse and human platelets and the initiation of neuroinflammation in the EAE model.26 Thus, we compared the level of 5HT release in response to LR from platelets of patients with MS versus HC (Figure 1D, right, induced serotonin release). We found that platelets from HC released serotonin in response to LR leading to 3.8-fold higher 5HT level in platelet-free plasma when compared with background levels of 5HT in plasma (Figure 1D; HC and HC-LR). At the same time, platelets from patients with MS did not have increased levels of 5HT release above background level in response to LR (Figure 1D, MS and MS-LR). Thus, in contrast to platelets from HC, platelets from patients with MS failed to secrete 5HT on stimulation with LR.
Unresponsiveness of platelets from patients with MS to LR could be because of desensitization of platelet signaling pathways or due to exhaustion or depletion of substances stored in platelet granules (such as 5HT) because of chronic activation. We found that platelets from patients with MS, which secreted lower amounts of 5HT in response to stimulation with LR, had a comparable level of activation of downstream signal pathways as measured by Ca2+ influx (Figure 1E, HC and MS), demonstrating that the activation of signaling pathways in platelets from patients with MS was unaltered. At the same time, we found that 5HT was substantially decreased in platelets from patients with MS (Figure 1C), which confirms the previously reported study.37 Thus, all of these results support the hypothesis that platelets become exhausted during MS in their ability to secrete 5HT, which will be further addressed. In summary, platelets from patients with MS formed aggregates with CD4 T cells, demonstrated signs of degranulation (annexin-V staining, formation of platelet-derived microparticles, and decrease in 5HT content in platelets), and failed to release 5HT in response to LR.
Platelets Become Annexin-V Positive During Early Stages of MS But Form Platelet–CD4 T-Cell Aggregates During Advanced Stages of the Disease
After the results showing broad distributions of percentages of platelet–CD4 T-cell aggregates in the peripheral blood of patients with MS (Figure 1A), we decided to compare HC with 3 groups of MS patients with different expanded disability status scale (EDSS) levels (these serve as an indicator of the progression of the disease) for platelet–CD4 T-cell aggregate formation. We also investigated the level of annexin-V staining in these 3 groups of patients with MS. The group of MS patients with more advanced stages of the disease (with the highest EDSS level range of 3–6.5) had the highest level of platelet–CD4 T-cell aggregates when compared with HC or 2 other groups of MS patients with lower EDSS levels (Figure 1F). On the other hand, we found that the highest level of annexin-V staining was observed in 2 groups of MS patients with early stages of the disease, with no or mild neurological symptoms (EDSS level ranges of 0–1 and 1.5–2.5, respectively), when compared with HC or the third group of patients with the highest EDSS level (range of 3–6.5; Figure 1G). Thus, platelets become annexin-V positive in MS patients with no or mild neurological symptoms, whereas the formation of platelet–CD4 T-cell aggregates was associated with progression of neurological clinical symptoms during more advanced stages of the disease.
Platelets From Patients With MS Are Exhausted in Their Ability to Release Contents From Both Dense and α-Granules in Response to Activating Stimuli
We have found previously that during stimulation of mouse and human platelets with LRs both dense granule component 5HT and α-granule components PF4 and IL-1α were released,26 and CD62P was upregulated (not shown). The result of action of LR on many features of platelet activation and degranulation was comparable with that of thrombin.26 Thus, we hypothesized that we observe general phenomena of exhaustion of platelets from patients with MS, which is characterized by the inability to secrete 5HT and PF4 (ie, both dense and α-granule components) in response to various stimuli, such as LR, thrombin, or ADP. To test this, we analyzed 3 HC individuals and 3 MS patients with impaired release of 5HT induced by LR and investigated PF4 release from the same individuals during the same experiment. Similar to our previous study,26 we found that platelets from HC released both 5HT and PF4. The releases of PF4 from HC platelets were less potent when compared with 5HT but were observed for HC platelets stimulated with LR or ADP (Online Table II). At the same time, platelets from patients with MS failed to release both 5HT and PF4 in response to LR or ADP (Online Table II). Thus, we found that platelets from patients with MS failed to release products from both dense and α-granules on stimulation with LR.
Normal Platelets Skew the Differentiation of CD4 T Cells Toward Th1 and Th17 Phenotypes
To investigate whether platelets affect the function of CD4 T cells, we performed coculture experiments of CD4 T cells with platelets isolated from the same healthy subjects. We found that at optimal physiological CD4 T cell/platelet ratio (1:15), platelets increased the percentages of IFN-γ (Figure 2A) and IL-17 (Figure 2B) producing CD4 T cells. Coculture of CD4 T cells with platelets did not result in an increase of regulatory T cells or Th2 cells: it was trend for decreased percentages of regulatory T cells (Figure 2C) and Th2 cells (Figure 2D). When we investigated the production of Th2- (IL-4), Th1- (IFN-γ and granulocyte-macrophage colony-stimulating factor [CSF]), and Th17- (IL-17 and granulocyte-macrophage CSF) associated cytokines in culture supernatants by ELISA, we found that coculture of CD4 T cells with platelets resulted in upregulation of Th1- and Th17-associated cytokines IFN-γ, IL-17, and granulocyte-macrophage CSF but not Th2-cytokine IL-4 (Figure 2E–H). Thus, coculture of human platelets with CD4 T cells promoted differentiation of human CD4 T cells toward Th1 and Th17 phenotypes.
Activated Platelets From Patients With MS Fail to Differentiate CD4 T Cells Toward Th1 and Th17 Phenotypes
We next compared the ability of platelets isolated from patients with MS or HC to skew the differentiation of CD4 T cells toward Th1 and Th17 phenotypes. We found that MS platelets had an impaired ability to skew the differentiation of CD4 T cells toward Th1 (Figure 3A, platelets), Th17 (Figure 3B, platelets), and IFN-γ/IL-17 double-positive CD4 T cells (Figure 3C, platelets) when compared with HC platelets. Moreover, platelets from patients with MS had impaired ability to stimulate proliferation of CD4 T cells and resulted in decrease in absolute numbers of Th1 and T17 cells in cocultures (not shown). In addition, CD4 T cells from patients with MS were less able to become differentiated toward Th1, Th17, and IFN-γ/IL-17 double-positive CD4 T cells when compared with HC (Figure 3A–3C, CD4).
In keeping with the observation that platelets from patients with MS were more activated ex vivo compared with those from HC (Figure 1A and 1B), we hypothesized that chronic in vivo activation and exhaustion of secretary granules lead to a decrease in the ability of platelets from patients with MS to activate CD4 T cells in vitro (Figure 3A–3C). To test this hypothesis, we activated in vitro platelets from HC with classic platelet-activating agonists thrombin or ADP (or incubated platelets with buffer as a control), washed them, and added them to cocultures. It was found that preactivation of HC platelets with thrombin, or ADP, substantially reduced the ability of platelets to enhance the production of IFN-γ (Figure 3D), IL-17 (Figure 3E), or both IFN-γ and IL-17 (Figure 3F) when cocultured with CD4 T cells. These results suggest that activated platelets lose their ability to stimulate Th1/Th17 differentiation.
Taken together, these data suggest that platelets from patients with MS are much less potent in skewing differentiation of CD4 T cells toward Th1 and Th17, which correlates with their activation and decrease in the ability to secrete proinflammatory factors, such as 5HT (Figure 1C).
Platelet-Derived Soluble Factors 5HT, PF4, and PAF Contribute to Th1/Th17 Differentiation
It was reported that platelet-derived soluble factors 5HT, PF4, and PAF could contribute to T-cell proliferation and Th1/Th17 differentiation.6,12,38–41 By using anti-5HT and anti-PF4 antibodies, and PAF receptor inhibitor, we found that platelet-derived 5HT contributed to increased proliferation of CD4 T cells and differentiation toward Th1, whereas PF4 and PAF contributed to Th17 differentiation (Online Figure II).
Activated Platelets Have High Capacity to Form Platelet–CD4 T Cell Aggregates, Which Leads to Decrease in CD4 T-Cell Proliferation and IFN-γ Production
In the previous experiments, we found that platelets became activated and the levels of platelet–CD4 T-cell aggregates were increased during MS (Figure 1A and 1B). Therefore, we investigated the role of activated versus not activated platelets in the formation of platelet–CD4 T-cell aggregates and the influence of aggregation of CD4 T cells and platelets on T-cell activation, proliferation, and differentiation. We hypothesized that activated platelets had a higher capacity to form aggregates with T cells and downmodulate T-cell activation in these aggregates. Two following findings supported our hypothesis. First, we found that human CD4 T cells that were aggregated with platelets had ≈10-fold lower levels of proliferation and ≈3-fold lower levels of IFN-γ production, when compared with CD4 T cells that were not aggregated with platelets in the same cocultures (Online Figure IIIA). Second, we observed that human thrombin-activated platelets had higher capacity to do both downmodulate CD4 T-cell proliferation and IFN-γ production in platelet–T cell-aggregates (Online Figure IIIB and IIIC) and form these platelet–CD4 T-cell aggregates (Online Figure IIID and IIIE). Thus, the formation of aggregates of human CD4 T cells with activated platelets resulted in downmodulation of T-cell proliferation and differentiation.
Platelets Promote Proliferation and Differentiation of MOG-Specific Autoimmune T Cells Toward Th1 and Th17 In Vivo
To confirm our in vitro results with human CD4 T cells, we used MOG T-cell receptor transgenic mice where ≈90% of T cells recognize myelin self-antigen MOG. Previously, we found that depletion of platelets substantially ameliorated EAE induced by active immunization of mice with MOG.26 In this study, we investigated the role of platelets in activation of MOG-specific T cells in vivo. We immunized MOG T-cell receptor transgenic mice with MOG35–55 peptide and found that depletion of platelets in vivo resulted in decreased proliferation of myelin-specific CD4 T cells as indicated by bromodeoxyuridine and 3H-thymidin incorporation assays (Figure 4A and 4B) and decrease in the percentages and absolute numbers of and IFN-γ– and IL-17–producing CD4 T cells (Figure 4C–4E). Furthermore, depletion of platelets increased apoptosis of myelin-specific CD4 T cells (Figure 4F), suggesting that platelets also provide survival factors to pathogenic T cells during their activation. Thus, we found that platelets contributed to the priming and activation of myelin-specific T cells in vivo and their differentiation toward Th1 and Th17.
Platelets Become Annexin-V Positive Early in EAE and Form Platelet–CD4 T-Cell Aggregates During Advanced Stages of the Disease
We further investigated annexin-V expression on platelets during EAE after our findings in MS (Figure 1B). Similar to patients with MS, we found that platelets from mice with EAE had 2.1-fold increase in the level of annexin-V staining (Figure 5A). This was observed during the preclinical stages of the disease (Figure 5A, day 5 and day 9). Starting from day 14 (EAE onset), the level of annexin-V staining returned to control levels of unmanipulated healthy animals (Figure 5A, day 14 and day 20), indicating that the process of platelet activation/degranulation was transient and occurred during the early stages of EAE. Because it is known that platelet activation/degranulation was accompanied by the release of platelets’ granule constituents, we investigated the level of 5HT in the mouse platelets activated in vitro with thrombin or ADP or investigated the 5HT level in platelets during EAE. We found that thrombin- and ADP-activated platelets had significantly reduced 5HT content (Figure 5B). We also found that the level of 5HT in platelets was decreased during EAE as early as day 5 post immunization. Serotonin content in the platelets reached its minimum level on day 9; after that, it gradually increased on days 14, 20, and 30 but still stayed below the 5HT level in platelets of healthy mice (Figure 5C). In contrast to the level of annexin-V staining, the relative level of platelet–CD4 T-cell aggregates began to increase on day 14 (disease onset) and was increased 2.3-fold during the peak of disease on day 20 (Figure 5D). Thus, platelet activation/degranulation occurred in early preclinical stages of the disease, whereas the formation of platelet–CD4 T-cell aggregates occurred during the peak of the disease.
CD62P Expression on Activated Platelets Plays an Important Role in the Formation of Platelet–CD4 T-Cell Aggregates and Downmodulation of T-Cell Activation
On the basis of our previous results (Online Figure III), we proposed that the ability of platelets to form aggregates with CD4 T cells implies that the platelets are activated and express CD62P. We tested this hypothesis in the mouse model and found that activated mouse platelets had high capacity to bind to CD4 T cells via CD62P–activated leukocyte cell adhesion molecule contact interactions and interfere with the delivery of costimulatory signals from antigen-presenting cells to T cells leading to a decreased level of CD4 T-cell activation (Online Figure IV). Contact interactions of activated mouse platelets with CD4 T cells were also evident in scanning electron microscopic images (Online Figure V). Thus, activated mouse platelets preferentially formed platelet–CD4 T-cell aggregates and decreased CD4 T-cell activation in these aggregates.
Formation of Platelet–CD4 T-Cell Aggregates Resulted in Amelioration of EAE
In the previous in vitro experiments, we found that adhesion of activated platelets to CD4 T cells resulted in decreased level of T-cell activation (Online Figures III–V). In addition, we demonstrated that anti-CD62P antibodies significantly decreased the formation of platelet–CD4 T-cell aggregates in vitro (Online Figure IV). It was reported that CD62P, which is expressed on activated platelets, played an important role in the formation of platelet–leukocyte aggregates, including CD4 T cells in humans and mice.42–46 On the basis of this, we decided to investigate the role of platelet–CD4 T-cell aggregates in vivo in the EAE model. We further investigated the level of platelet–CD4 aggregates starting from the peak of the disease at day 20 when we initially observed an increase in the level of platelet–CD4 T-cell aggregates (Figure 5D). We found that the percentages of platelet–CD4 T-cell aggregates in the peripheral blood reached the highest level on day 35, when mice spontaneously recovered from the disease (Figure 6A and 6B, control serum). When we isolated mononuclear cells from the spleen using the Ficoll gradient, we also found that platelets formed aggregates with CD4 T cells in the spleen during EAE with kinetics similar to that of peripheral blood (Figure 6C, control serum). When we injected anti-CD62P antibodies intravenously starting from day 20 as indicated by arrows in Figure 6A, we found that the levels of platelet–CD4 T-cell aggregates were substantially decreased by day 35 in peripheral blood and spleen (Figure 6B and 6C; anti-CD62P), whereas EAE was substantially exacerbated when compared with the control group of mice (Figure 6A). As an alternative method to decrease the level of CD4 T-cell aggregates, we performed the depletion of platelets with antithrombocyte serum, as we did in Figure 4. Administration of antithrombocyte serum during EAE starting from day 20 also resulted in a decrease in platelet–CD4 aggregates in peripheral blood and spleen, and the effect was similar to that of anti-CD62P antibodies (Figure 6B and 6C, antithrombocyte serum). In addition to exacerbation of EAE (Figure 6A), administration of anti-CD62P antibodies resulted in ≈2-fold and ≈4-fold increases in the level of proliferation of CD4 T cells in the spleen and CNS on day 35, respectively (Figure 6D–6G). Thus, we found that formation of platelet–CD4 T-cell aggregates in vivo decreased the level of proliferation of CD4 T cells in the CNS and periphery and contributed to recovery from EAE.
Depletion of Platelets During Late Stages of EAE Exacerbates the Disease
Because the formation of platelet–CD4 T-cell aggregates resulted in milder EAE disease symptoms (Figure 6A) and depletion of platelets decreased the percentage of platelet–CD4 T-cell aggregates in peripheral blood and spleen (Figure 6B and 6C), we further investigated the role of platelets during the late stages of EAE by performing platelet depletion. We have previously found that during preclinical stages of the disease, the depletion of platelets substantially ameliorated EAE,26 which was consistent with the present study demonstrating that normal (not activated) platelets stimulated Th1/Th17 cells (Figures 2 and 4). Here, we performed the depletion of platelets during the progression of EAE. We found that depletion of platelets starting from peak of EAE (day 20) exacerbated the disease (Figure 7A), resulting in an enhanced level of CNS inflammation, as determined by the level of infiltration of macrophages, lymphocytes, and CD4 T cells into the CNS (Figure 7B and 7C), and enhanced CD4 T-cell proliferation levels in the CNS (Figure 7D and 7E) and periphery (Figure 7F and 7G) on day 35. Similar to our observation for human CD4 T cells (Online Figure III), we found that mouse CD4 T cells that were aggregated with platelets had 18-fold lower level of proliferation and 3.5-fold lower level of IFN-γ production during EAE (day 35) when compared with CD4 T cells that were not aggregated with platelets (Online Figure VI). Thus, we further demonstrated that platelets play a regulatory role during progression of EAE and most likely during progression of MS.
In this article, we have demonstrated a dual role for platelets in the stimulation and inhibition of pathogenic CD4 T cells in MS. Normal (not activated) but not in vitro or in vivo activated platelets produced 5HT on stimulation and contributed to Th1/Th17 differentiation. In both patients with MS and the EAE model, signs of platelet activation/degranulation were observed during the early stages of the disease, whereas the levels of platelet–CD4 T-cell aggregates were increased during the progression of the disease at more advance stages. Activated platelets had higher capacity to form platelet–CD4 T-cell aggregates during disease progression, and adhesion of activated platelets to CD4 T cells decreased T-cell activation leading to downmodulation of EAE.
Serotonin is an important neurotransmitter that can directly affect the functions of neurons in the CNS and can modulate the functions of the immune cells in the periphery, and thus, it may play a role in MS epidemiology.47 In mammals, 5HT is found in the gastrointestinal tract, platelets, and in the CNS.38 In the peripheral blood, platelets are the exclusive source of serotonin that is stored in platelet granules.3,8 It has been reported that elevated levels of 5HT in the CSF correlated with MS progression and might thus indicate platelet degranulation and secretion of 5HT in the CNS in the areas of inflammatory lesions on recognition of LR.26 This was supported by the finding that during EAE and MS, other platelet-derived factor PAF accumulated in the CNS and CSF.48,49 These platelet-derived soluble factors could promote Th1/Th17 differentiation in the CNS and periphery. During advanced stages of the disease, secretory granules become depleted/exhausted and activated platelets form aggregates with T cells (Online Figure V) and suppress them. At the final stage of platelet exhaustion, it is possible that activated platelets shed their surface receptors, such as CD62P, on budding microparticles (Online Figures I and V) and stop interacting with T cells. The inhibitory effect of activated platelets was recently demonstrated for rheumatoid arthritis. It was shown for this disease that adhesion of platelets to CD4 T cells led to a decrease in their proliferation and expression of IL-17 and IFN-γ and an increase in IL-10.10
Thus, this study suggests that degranulation and subsequent release of several platelet-derived soluble factors contributed to CD4 T-cell proliferation and proinflammatory cytokine production, whereas formation of aggregates with platelets resulted in decrease in T-cell proliferation and production of proinflammatory cytokines. Thus, depending on the stage of MS or EAE disease, platelets could play proinflammatory and regulatory roles as summarized in Figure 8. In early stages of MS, platelets have high levels of proinflammatory mediators stored in their granules, such as 5HT, PAF, and PF4, which are released when platelets become activated at the site of CNS inflammation by recognizing brain-specific LR or subendothelial matrix in damaged blood vessels (Figure 8A). In addition, degranulating platelets could produce many mediators that activate endothelial cells and promote migration of CD4 T cells into the CNS (Figure 8A). Late in MS, the granule constituents of activated platelets become depleted, but platelets upregulate CD62P and adhere to activated CD4 T cells leading to their deactivation (Figure 8B).
We found in our study that the formation of platelet–CD4 T-cell aggregates was mediated at least in part via CD62P on activated platelets and CD166 on activated CD4 T cells. Besides CD166, CD62P on platelets can bind to other molecules on CD4 T cells, such as P-selectin glycoprotein ligand-142 and TIM-1 (T-cell immunoglobulin and mucin domain 1).50 In addition, activated platelets express many integrins that could also contribute to cell contact interactions of platelets with activated T cells.51 We think that binding of platelets to T cells, antigen presenting cells, and activated endothelial cells resulted in perturbed interaction of CD4 T cells with antigen-presenting cells and endothelial cells to cause decrease in T-cell activation and migration of T cells to the site of inflammation (Figure 8B). In support of our hypothesis, it was reported that activated leukocyte cell adhesion molecule substantially contributed to human and mouse T-cell activation and migration of CD4 T cells into the CNS in the EAE model.52,53
In summary, our data indicate a role for platelets in the immunopathogenesis of CNS autoimmune demyelinating disease, which may provide new opportunities for diagnostics and the treatment of MS by monitoring and modulating the functions of platelets.
Sources of Funding
This work was supported by National Institutes of Health RO1 NS071039 research grant (United States), the LCWIIM (Lui Che Woo Institute of Innovative Medicine: BRAIN Theme) fund (Hong Kong), the LKS (Lo Kwee-Seong) Biomedical Research fund (Chinese University of Hong Kong), and HMRF (Health and Medical Research Fund) grant reference no. 02130636 (Hong Kong).
In July 2015, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.38 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.115.306847/-/DC1.
- Nonstandard Abbreviations and Acronyms
- 5-hydroxytryptamine (serotonin)
- colony-stimulating factor
- experimental autoimmune encephalitis
- healthy control
- lipid raft
- myelin oligodendrocyte glycoprotein
- multiple sclerosis
- platelet-activating factor
- platelet factor 4
- T helper (CD4 T cell)
- Received May 18, 2015.
- Revision received August 18, 2015.
- Accepted August 20, 2015.
- © 2015 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Platelets can modulate inflammation by producing proinflammatory factors and interacting with immune cells.
Platelets become activated during autoimmune diseases, such as multiple sclerosis, but the role of these cells in the pathogenesis of autoimmune diseases remains uncertain.
Although experimental autoimmune encephalitis is widely used as a model for multiple sclerosis, not all results obtained in the experimental autoimmune encephalitis model could be directly translated to multiple sclerosis.
What New Information Does This Article Contribute?
In early stages of multiple sclerosis and experimental autoimmune encephalitis, platelets become activated and produced soluble proinflammatory factors that specifically stimulated the differentiation of CD4 T cells toward pathogenic T helper-1 and T helper-17 phenotypes and promoted neuroinflammation.
At the more advanced stages of neuroinflammation, activated platelets became exhausted in their ability to secrete proinflammatory factors but substantially increased their ability to form aggregates with CD4 T cells, contributing to downmodulation of T-cell activation and resolution of inflammation.
The results obtained with the experimental autoimmune encephalitis model are in a good agreement with the results in patients with multiple sclerosis.
It is known that platelets regulate inflammation in many types of pathological conditions; however, the exact mechanism of such regulation remains uncertain. We found that at early stages of central nervous system autoimmune inflammation (also known as neuroinflammation), platelets stimulate adaptive immune response by promoting the expansion and differentiation of CD4 T cells toward T helper-1 and T helper-17 phenotypes, which play a pathogenic role in several types of autoimmune diseases, including multiple sclerosis. During resolution of neuroinflammation, activated platelets form aggregates with CD4 T cells leading to a decrease in T-cell activation, which contributed to recovery from the disease. These results suggest that platelets play an important role in the regulation of adaptive immune response during inflammation, which might be viewed as a general mechanism by which platelets regulate immune responses during inflammatory diseases. Under such pathological conditions, platelets could be considered as innate-like immune cells that stimulate or inhibit CD4 T cells during initiation and resolution of inflammation. Hence, investigation of platelet–CD4 T-cell interactions may have implications for diagnostics and treatment of several types of pathological inflammatory conditions associated with neurovascular diseases, such as multiple sclerosis, stroke, and atherosclerosis.