Interleukin 1 receptor 1 and interleukin 1β regulate megakaryocyte maturation, platelet activation, and transcript profile during inflammation in mice and humans

Abstract

Objective: Interleukin 1 Receptor 1 (IL1R1) and its ligand, IL1β, are upregulated in cardiovascular disease, obesity, and infection. Previously, we reported a higher level of IL1R1 transcripts in platelets from obese individuals of the Framingham Heart Study (FHS), but its functional effect in platelets has never been described. Additionally, IL1β levels are increased in atherosclerotic plaques and in bacterial infections. The aim of this work is to determine whether IL1β, through IL1R1, can activate platelets and megakaryocytes to promote atherothrombosis.

Approach and results: We found that IL1β-related genes from platelets, as measured in 1819 FHS participants, were associated with increased body mass index, and a direct relationship was shown in wild-type mice fed a high-fat diet. Mechanistically, IL1β activated nuclear factor-κB and mitogen-activated protein kinase signaling pathways in megakaryocytes. IL1β, through IL1R1, increased ploidy of megakaryocytes to 64+ N by 2-fold over control. IL1β increased agonist-induced platelet aggregation by 1.2-fold with thrombin and 4.2-fold with collagen. IL1β increased adhesion to both collagen and fibrinogen, and heterotypic aggregation by 1.9-fold over resting. High fat diet-enhanced platelet adhesion was absent in IL1R1(-/-) mice. Wild-type mice infected with Porphyromonas gingivalis had circulating heterotypic aggregates (1.5-fold more than control at 24 hours and 6.2-fold more at 6 weeks) that were absent in infected IL1R1(-/-) and IL1β(-/-) mice.

Conclusions: In summary, IL1R1- and IL1β-related transcripts are elevated in the setting of obesity. IL1R1/IL1β augment both megakaryocyte and platelet functions, thereby promoting a prothrombotic environment during infection and obesity; potentially contributing to the development of atherothrombotic disease.

Keywords: IL1R1 protein, human; blood platelets; diet, high-fat; infection; megakaryocytes.

Figure 1

Expression of interleukin (IL)1R1 and function on megakaryocytes. Representative histogram of Meg-01 cells (A) stained with anti-human IL1R1 fluorescein isothiocyanate (FITC); n=5; IC indicates isotype control. B, Representative Western blots probed for phospho- and total NFκB p65, phospho- and total ERK, and phospho- and total p38 in Meg-01 cells. C, Quantification of the increases in phosphorylation of NFκB p65, ERK, and p38 after treatment with IL1β. n=4; *P<0.05, **P<0.01 compared with NT. D, Representative Western blots probed for phospho- and total NFκB p65, phospho- and total Akt, phospho-and total ERK, and phospho- and total p38 in isolated wild-type (WT) and IL1R1^−/− mouse megakaryocytes; n=4. Isolated WT (E) or IL1R1^−/− (F) mouse bone marrow cells were treated with vehicle (Control – Ctrl), thrombopoietin (TPO), or IL1β for 3 days. Megakaryocyte DNA content (2 N-64+N) was measured. n=18; *P<0.05, **P<0.01 compared with Control, #P<0.05, ##P<0.01 compared with TPO.

Figure 2

Interleukin (IL)1β alters megakaryocyte inflammatory- and thrombosis-related gene expression through NFκB, ERK, and PI3K/ Akt pathways. Gene expression of Meg-01 cells pretreated with 50 μmol/L LY294002, 50 μmol/L U0126, 50 μmol/L BAY 11–7082, or dimethyl sulfoxide (DMSO), then treated with IL1β. COX2 (A; n=8), monocyte chemotactic protein-1 (MCP-1; B; n=5), NFκB1 (C; n=8), IL1R1 (D; n=3), IL1β (E; n=4), TLR2 (F; n=7), CD41 (G; n=10), and GP1b (H; n=7). *P<0.05, **P<0.01, ***P<0.001 compared with NT (No Treatment). Gene expression in isolated wild-type (WT; I) and IL1R1^−/− (J) mouse megakaryocytes treated with IL1β. n=3 for WT; n=3 for IL1R1^−/−. *P<0.05, **P<0.01, ***P<0.001 compared with NT.

Figure 3

Interleukin (IL)1β affects platelet function. A, Representative histogram of isolated human platelets stained with anti-human IL1R1 FITC antibodies; n=5; IC indicates isotype control. B, Isolated human platelets aggregation was measured for 10 minutes in the presence of collagen. n=4; **P<0.01 compared with collagen alone. C, Isolated human platelet aggregation was measured for 10 minutes in the presence of increasing concentrations of thrombin. n=10; ***P<0.001, *P<0.05 compared with thrombin alone. Isolated WT (D; n=4) and IL1R1^−/− (E; n=3) mouse platelet aggregation was measured in the presence of thrombin. ***P<0.001 compared with thrombin alone. F, Isolated human platelet aggregation was measured in the presence of thrombin for 10 minutes. n=15; ***P<0.001 compared with thrombin alone. G, Representative isolated human platelets treated with IL1β or thrombin, or IL1β, then thrombin adherent on collagen or fibrinogen; n=3. H, The percentage of platelet-positive neutrophils in the presence of Pam3CSK4 (PAM) or IL1β. n=6; *P<0.05, **P<0.01 compared with Resting.

Figure 4

Interleukin (IL)1β affects adhesion in the setting of high fat diet. A, Representative photographs of isolated wild-type (WT) and IL1R1^−/− platelets treated with IL1β, thrombin, or both and adherent on collagen at baseline; n=5 to 6 mice in each group. B, Representative photographs of isolated WT and IL1R1^−/− platelets treated with thrombin and adherent to collagen; n=3 to 4 mice in each group.

Figure 5

Platelet RNA expression in Framingham Heart Study (FHS) individuals associated with increased body mass index (BMI). The platelet RNA expression of IL1β-associated genes in FHS individuals stratified by BMI status adjusted by clinical covariates listed in Tables III and IV in the online-only Data Supplement. Relative expression values (ΔCt) corrected by the housekeeping genes (GAPDH, B2M, α-actin [ACTB]) were rescored so that larger values represent increased expression. MMP9, NLRP3, and myeloid differentiation primary response gene (MYD88) were all significantly increased in obese individuals (BMI≥30) compared with normal weight individuals (BMI<25); *P<0.05.

Figure 6

Schematic of the effects of interleukin (IL)1β/IL1R1 on megakaryocyte and platelet function. A, A high fat diet will cause megakaryocytes to produce platelets with an increase in both inflammatory and thrombotic genes. Red highlighted data represent new Framingham Heart Study (FHS); green highlighted data represent mouse data; *confirmed in both human and mouse platelets; Bolded genes were confirmed to be upregulated in Meg-01 by IL1β. B, IL1β in circulation as a result of increased body weight will bind IL1R1 on megakaryocytes. This interaction leads to the activation of the nuclear factor (NF)κB, PI3K/Akt, and mitogen activated protein kinase (MAPK) (ERK and p38) pathways. As a result, there is an increase in megakarycoyte maturation, including increased adhesion, increases in ploidy, and increases in mRNA production of inflammatory and thrombotic genes. IL1β can also bind IL1R1 on platelets and either enhance aggregation induced by agonists or promote adhesion and heterotypic aggregate formation.