Platelets Play Differential Role During the Initiation and Progression of Autoimmune Neuroinflammation

Abstract

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.

Keywords: T-lymphocytes; blood platelets; helper-inducer; interleukin-17; multiple sclerosis; serotonin.

© 2015 American Heart Association, Inc.

Figures

Figure 1. Analysis of platelet-CD4 T cell aggregates, expression of Annexin V on platelets, serotonin content in platelets, and serotonin secretion by platelets isolated from peripheral blood of MS patients or healthy controls (HC)

(A) Flow cytometry analysis of platelet-CD4 T cell aggregates. Whole blood preparations were stained for CD4 and the platelet marker CD42a and percentages of platelet-CD4 T cell aggregates were determined as co-expression of CD42a and CD4 of all CD4+ T cells. Percentages of CD42a+CD4+ T cells aggregates are shown for each individual. (B) Flow cytometry analysis of platelet surface staining for Annexin V on CD42a+ platelets from platelet rich plasma. The expression of Annexin V on CD42a+ gated platelets isolated from patients with untreated MS vs. healthy control subjects is presented as mean fluorescence intensity (MFI). (C) Comparison of the serotonin content in the platelets of HC vs. MS patients. Platelet rich plasma was obtained from the peripheral blood, platelets were counted and the volume was adjusted to have equal number of platelets in each sample. After that the platelets were isolated by centrifugation and the amount of serotonin was determined in platelet lysate as described in Methods. (D) Platelets were isolated from peripheral blood of HC or MS patients and the amount of spontaneous or brain lipid raft (LR)-induced serotonin release was measured in platelet-free plasma by ELISA as described in Methods. (E) Comparison of platelet Ca2+ influx in response to brain lipid rafts (LR) for platelets from MS patients (low serotonin producers in response to LR) vs. healthy control subjects (high serotonin producers in response to LR). One representative experiment of three is shown. The changes in the level of intracellular Ca2+ (y-axis) vs. time (x-axis) was assessed using fluorescent probe Fura 2AM as described in Methods.(F–G) Comparision of percentages of platelet-CD4 T cell aggregates (F) and Annexin V expression on platelets (G) in the peripheral blood of HC vs. three groups of MS patients with 1) low expanded disability status scale (EDSS) range (0–1), 2) intermediate EDSS range (1.5–2.5), and 3) high EDSS range (3–6.5). In (A–D, F–G), mean with individual data points or mean ± S.E. is shown (*, p<0.05; **, p<0.01; ***, p<0.005). Unpaired Student’s tests and Mann–Whitney U tests were used to determine statistical significance as described in Methods.

Figure 2. Co-culture of human platelets with human CD4 T cells increases percentages of Th1 and Th17 cells and production of Th1- and Th17- associated cytokines, IFN-γ, IL-17 and GM-CSF

Human CD4 T cells were isolated from PBMCs of healthy control (HC) subjects by negative selection. CD4 T cells were stimulated with anti-CD3 (1µg/ml) and anti-CD28 (1µg/ml) antibodies and cultured alone or co-cultured with various numbers of syngeneic platelets (CD4 T cell/platelet ratio 1:15, 1:1.5 and 1:0.15) for six days (see Methods). (A–B) Percentages of IFN-γ (A) and IL-17 (B) producing CD4 T cells were analyzed by flow cytometry using surface staining for CD4 and intracellular staining for IFN-γ and IL-17 as described in Methods. (C) Percentages of CD25hiFoxP3+CD4+ Tregs were determined using multicolor flow cytometry as described in Methods. (D) Percentages of IL-4+CD4+ Th2 cells were determined by flow cytometry as described in Methods. (E–H) The production of Th2-(IL-4 (E)), Th1- (IFN-γ (F), GM-CSF (H)) and Th17- (IL-17 (G), GM-CSF (H)) associated cytokines in culture supernatants were determined by ELISA (see Methods). In (A–H), mean ± S.E. of 3–5 separate experiments is shown (*, p<0.05; **, p<0.01; ns, not significant).

Figure 3. Comparison of the ability of platelets from MS patients vs. healthy control (HC) subjects, or activated vs. non-activated platelets, to affect differentiation of CD4 T cells towards Th1 and Th17 phenotypes

(A–C) Human CD4 T cells from healthy controls or MS patients were co-cultured with syngeneic platelets or with allogeneic platelets from MS or HC at ratio of 1:15 and the percentages of Th1 (A), Th17 (B), and IL-17/IFN-γ double-positive CD4 T cells (C) were determined by flow cytometry as described in Methods. (D–F) Human CD4 T cells from HC were cultured alone or co-cultured with syngeneic platelets as for Fig. 2 at 1:15 CD4 T cell/platelet ratio. Platelets were pre-incubated with buffer, thrombin (TB; 0.1 U/ml) or ADP (500 µM) for 30 min, washed, then added to CD4 T cells for co-culture. After six days of co-culture, the percentages of IFN-γ (C), or IL-17 (D), or IL-17/IFN-γ (F) producing CD4 T cells were determined by flow cytometry as described in Methods. In (A–F), mean ± S.E. of 4–5 separate experiments is shown (*, p<0.05).

Figure 4. Effect of platelet depletion on proliferation, Th1/Th17 differentiation, and cell death of myelin-specific autoimmune CD4 T cells in vivo

(A–F) MOG-TCR transgenic mice were immunized with MOG35–55 peptide and CFA as described in Methods. On days 0, 2, 4, 6, and 8 post-immunization 30 µl of anti-thrombocyte (anti-TS) or control serum (CS) was injected i.p., and on day 10 post-immunization splenic CD4 T cells were restimulated in vitro with MOG35–55 peptide as described in Methods. (A–B) CD4 T cell proliferation was assessed on d3 after restimulation with MOG35–55 peptide (2–10 µg/ml) by BrdU (A) and 3H-Td (B) incorporation assays. In (A), percentages of BrdU+ CD4 T cells are shown. (C–F) On day 10 post-immunization splenic and draining lymph nodes CD4 T cells were collected and analyzed for expression of IFN-γ (C,E) and IL-17 (D,E) by intracellular staining using multicolour flow cytometry. Absolute numbers of IFN-γ and IL-17-producing CD4 T cells are shown in (E) (see Methods). (F) MOG TCR transgenic 2D2 mice were immunized with MOG/CFA. On day 0, 2, 4, 6 and 8 post-immunization 30 µl of anti-TS or CS was injected i.p., and on day 10 post-immunization splenic CD4 T cells were analyzed by flow cytometry for the expression of Annexin V (AnnV, apoptosis marker) and plasma membrane permeability using 7-Aminoactinomycine D (7AAD, necrosis marker, see Methods). Percentages of AnnV+7AAD− (early apoptosis), AnnV+7AAD+ (late apoptosis) and AnnV−7AAD+ (necrosis) cells of all CD4 T cells are shown. In (A, C–F), mean ± S.E. of 6–8 individual mice is shown (*p<0.05; **, p<0.01). In (B) mean ± S.E. of triplicate is shown. Effectiveness of platelet depletion by i.p. administration of anti-TS was assessed by platelet counts in the peripheral blood as described in Methods.

Figure 5. The role of platelets in the pathogenesis of EAE: expression of Annexin V, serotonin level in platelets and CD4 T cell-platelet aggregate formation at different stages of the disease

Relative level of expression of platelet Annexin V on CD41+CD61+ gated mouse platelets (A), serotonin content in mouse platelets (B–C), and percentages of platelet-CD4 T cell aggregates (D) in the peripheral blood of unmanipulated (Naïve) B6 mice, mice immunized with CFA and i.p. Pertussis toxin (CFA), or MOG/CFA and i.p. Pertussis toxin (EAE). In (B–C), platelet rich plasma was obtained from mouse peripheral blood, and platelets were isolated from platelet rich plasma of normal mice (B, C) or mice with EAE (C) by centrifugation, and the amount of serotonin was determined in platelet lysate as described in Methods. In (B), washed platelets were activated with TB or ADP in vitro as described in Methods and then level of 5HT was analyzed in platelet lysate. Relative levels of 5HT are shown in comparison with unmanupulated mice. In (A–D), mean ± S.E. of total 4–10 individual mice for each group is shown for two separate experiments (*, p<0.05; **, p<0.01; ***, p<0.001).

Figure 6. The role of platelet-CD4 T cell aggregates in the pathogenesis of EAE

(A) EAE was induced and anti-CD62P antibodies (anti-CD62P) were injected i.v. starting from d20 as described in Methods. EAE clinical course (mean ± S.E.) of total 8–12 individual mice is shown for two separate experiments. Injection of anti-CD62P or CS is indicated by arrows. (B–C) The flow cytometry analysis of platelet-CD4 T cell aggregates in the peripheral blood (B) or spleen (C) of the healthy mice, or mice with EAE treated with anti-CD62P, anti-TS (see Methods) or CS as on d20, 22, 24, 27, 29, 31 and 33. The cells from the peripheral blood or spleen of healthy mice or mice with EAE on day 20 or day 35 after EAE induction were stained for CD4 and CD41 and analyzed by flow cytometry as described in Methods. The percentages of CD4+CD41+ platelet-CD4 T cell aggregates are shown. (D–G) Comparison of the levels of proliferation of CD4 T cells in the CNS (D, E) and spleen (F,G) of mice with EAE treated with anti-CD62P or CS on day 35 after disease induction. Proliferation assay was performed as described in Methods. Representative contour-plots with percentages of BrdU+ CD4 T cells are shown. Isotype-matched control staining for CD41 is shown in upper left corner of each contour-plot. In (B), (C), (E) and (G), mean ± S.E. of the group of 5–8 individual animals is shown (**, p<0.01; ***, p<0.005).

Figure 7. The role of platelets in the pathogenesis of EAE during late stages of the diseases

(A) EAE was induced and anti-thrombocyte (anti-TS) or control serum (CS) were injected i.p. starting from d20 as described in Methods. EAE clinical course (mean ± S.E.) of total 12–15 individual mice for each group is shown for three separate experiments. Injection of anti-TS or CS is indicated by arrows. (B–C) The flow cytometry analysis of the CNS infiltrating cells isolated from the mice with EAE treated with anti-TS or CS. The mononuclear cells were isolated from the CNS on day 35 after EAE induction, stained for CD11b and CD45, or CD3 and CD4, and analyzed by flow cytometry as described in Methods. The percentages of populations of CD11b+CD45low microglia (left gates), CD11b+CD45hi macrophages (upper right gates) and CD11b−CD45hi lymphocytes (lower right gates) are shown in (B). The quantification of the absolute number of macrophages, lymphocytes and CD4 T cells (CD3+CD4+) in the CNS is shown in (C) (see Methods). (D–G) Comparison of the levels of proliferation of CD4 T cells in the CNS (D, E) and spleen (F,G) of mice with EAE treated with anti-TS or CS on day 35 after disease induction. Proliferation assay was performed as described in Methods. Representative contour-plots with percentages of BrdU+ CD4 T cells are shown. Isotype-matched control staining for BrdU is shown in upper left corner of each contour-plot. In (C), (E) and (G), mean ± S.E. of the group of 5–8 individual animals is shown (*, p<0.05; **, p<0.01; ***, p<0.005).

Figure 8. Model of differential regulation of CNS autoimmune inflammation by platelets during disease initiation and progression

(A) In the early stages of the neuroinflammation platelets have non-activated phenotype with a low level of P-selectin (CD62P) expression and a high level of proinflammatory factors stored in platelet’s granules. During initial neurovascular damage in the early stages of CNS inflammation, normal platelets degranulate upon interaction with astroglial or neuronal glycolipids (brain lipid rafts), and/or subendothelal matrix, and the constituents of platelet granules PAF, PF4 and 5HT are released. In the CNS and the periphery platelet-derived PAF, PF4 and 5HT increased proliferation and differentiation of CD4 T cells into pathogenic Th1/Th17 cells upon interaction with antigen presenting cells (APCs). In addition, soluble platelet-derived mediators activated endothelial cells and facilitate the migration of CD4 T cells from blood vessels into the CNS. (B) In the late stages of the neuroinflammation platelets exhibited activated phenotype with low levels of platelet granule constituents such as 5HT and high levels of P-selectin (CD62) expression that resulted in adherence of platelets to activated CD4 T cells or APCs both of which express ALCAM. Formation of platelet-CD4 T cell aggregates prevented interaction of CD4 T cells with APCs in the CNS or periphery, leading to T cell deactivation and downmodulation of CNS inflammation. In addition, formation of platelet-CD4 T cell aggregates prevent migration of CD4 T cells into the CNS.