Thrombocytopenia Independently Leads to Changes in Monocyte Immune Function

Abstract

Background: While platelets have well-studied hemostatic functions, platelets are immune cells that circulate at the interface between the vascular wall and white blood cells. The physiological implications of these constant transient interactions are poorly understood. Activated platelets induce and amplify immune responses, but platelets may also maintain immune homeostasis in healthy conditions, including maintaining vascular integrity and T helper cell differentiation, meaning that platelets are central to both immune responses and immune quiescence. Clinical data have shown an association between low platelet counts (thrombocytopenia) and immune dysfunction in patients with sepsis and extracorporeal membrane oxygenation, further implicating platelets as more holistic immune regulators, but studies of platelet immune functions in nondisease contexts have had limited study.

Methods: We used in vivo models of thrombocytopenia and in vitro models of platelet and monocyte interactions, as well as RNA-seq and ATAC-seq (assay for transposase-accessible chromatin with sequencing), to mechanistically determine how resting platelet and monocyte interactions immune program monocytes.

Results: Circulating platelets and monocytes interact in a CD47-dependent manner to regulate monocyte metabolism, histone methylation, and gene expression. Resting platelet-monocyte interactions limit TLR (toll-like receptor) signaling responses in healthy conditions in an innate immune training-like manner. In both human patients with sepsis and mouse sepsis models, thrombocytopenia exacerbated monocyte immune dysfunction, including increased cytokine production.

Conclusions: Thrombocytopenia immune programs monocytes in a manner that may lead to immune dysfunction in the context of sepsis. This is the first demonstration that sterile, endogenous cell interactions between resting platelets and monocytes regulate monocyte metabolism and pathogen responses, demonstrating platelets to be immune rheostats in both health and disease.

Keywords: acute coronary syndrome; chromatin assembly and disassembly; monocytes; platelet count; toll-like receptor agonists.

Figure 1.

Thrombocytopenia alters circulating monocyte gene expression. A) Acute model of thrombocytopenia. PF4cre-DTRflox mice or WT control mice were treated with diphtheria toxin (DT) (400 ng/mouse) and platelet counts determined on multiple days post-DT. n=4 mice in each group. B) Circulating neutrophils, circulating and BM monocytes were isolated from control and thrombocytopenic mice and gene expression determined by RT-qPCR, normalized to Actb; n=independent neutrophils isolated from 4 mice; n=independent monocytes isolated from 6 mice. C) Isolated circulating monocytes from WT and thrombocytopenic mice were treated with LPS ex vivo and 24 hrs later, IL-6 and CXCL-1 were determined by ELISA; n=6 mice in each group. D-E) Circulating monocyte mRNA was isolated on d5 post-DT from control and thrombocytopenic mice for RNA-seq; n=3 in each group. D) GO analysis of up and down regulated genes indicated increased inflammatory and decreased metabolism gene expression in thrombocytopenic mouse monocytes, E) confirmed by RT-qPCR; n=6 mice in each group. F) Glycolytic rate assays were performed on circulating monocytes from WT and DT-induced thrombocytopenic mice; n=4 WT mice; n=8 thrombocytopenic mice. Data were represented as mean±SEM. Statistics: unpaired, 2-tailed Student’s t test in B (circulating and BM monocytes), C and E (Cxcl1, Il1b, Cxcl2, Pfkl, Tpi1, Ldha and Hif1a); Welch t test in E (Cxcl5); Mann-Whitney test in B (circulating neutrophils), E (Il6) and F.

Figure 2.

Resting platelets change monocyte gene expression in a contact dependent manner. A) Resting platelets limited monocyte and BMDM cytokine production in a platelet ratio dependent manner. Primary mouse BM monocytes, BMDMs or human CD14⁺CD16⁻ monocytes were co-incubated overnight with platelets at multiple platelet:monocyte ratios and cells then washed to remove platelets before LPS stimulation. IL-6 was quantified 24 hrs later; n=6 replicates from 3 mice; n=4 replicates from 1 human blood donor. B) Monocytes were cultured with platelets directly or separated in a transwell chamber overnight and monocytes then LPS stimulated; n=4 replicates from 2 mice. C-D) Monocytes were incubated overnight in control buffer or with platelets. Platelets were removed and mRNA isolated for RNA-seq or monocytes were LPS stimulated and 4 hrs later mRNA isolated for RNA-seq. C) GO analysis of up and down regulated genes in platelet cultured monocytes before LPS stimulation and D) confirmation of select metabolism and immune related genes after incubation in control or platelet co-culture conditions; n=6 replicates from 2 mice. E) CXCL-1 and CCL-2 production were determined in monocytes cultured with control media or platelets then LPS stimulated. n=3–4 replicates from 2 mice. F) Monocytes/BMDMs were cultured with control media or platelets overnight and platelets washed away before TLR2 agonist, TLR9 agonist or IL-1β stimulation. IL-6 was determined by ELISA 24hs later; n=4–6 replicates from 2 mice. Data were represented as mean±SEM. Statistics: One-way ANOVA followed by Tukey’s multiple comparisons in A (monocytes and BMDMs); Kruskal-Wallis followed by Conover-Iman post hoc test with Holm-sidak corrections in A (human monocytes) and B; Unpaired, 2-tailed Student’s t test in D and F (TLR2 and TLR9 agonists); Mann-Whitney test in E and F (IL-1β).

Figure 3.

Thrombocytopenia increased bacteria associated immune activation in vivo. A) Plasma IL-6 and IL-8 inversely correlated with platelet counts in septic patients. Plasma IL-6 and IL-8 were determined by ELISA and correlation to the platelet count was determined by linear regression followed by Pearson correlation analysis. n=12 individual septic blood donors. B) Circulating monocyte IL6 and IL8 inversely correlated with platelet counts in both septic and non-septic human patients. Monocyte IL6 and IL8 relative to the platelet counts were determined by linear regression followed by Pearson correlation analysis. n=20 (sepsis patient); n=7 (non-septic patient). C) Thrombocytopenic mice or WT control mice were treated with LPS (20 mg/kg) i.p. on d5 post-DT and 4 hrs later plasma IL-6, CXCL-1 and TNFα were determined by ELISA. n=6 mice in each group. D) Male and female thrombocytopenic mice and WT control mice were injected with cecal slurry (CS) i.p. (0.8 mg/g) and 4 hrs later plasma IL-6 and TNFα were determined by ELISA. n=5 mice in each group. E) Monocytes/macrophages were depleted with chlodronate liposomes on d4 and d5 post-DT and 4 hrs after the second dose, mice were LPS treated and 4 hrs later plasma IL-6 and TNFα were measured by ELISA. n=4 mice in each group. Data were represented as mean±SEM. Statistics: linear regression followed by Pearson correlation analysis in A and B; Unpaired, 2-tailed Student’s t test in C (IL6 and CXCL-1); Welch t test in C (TNFα); 2-way aligned rank transformation (ART) ANOVA with Sidak corrections in D and E.

Figure 4.

Platelet CD47 mediates monocyte immune programming. A) Monocytes were incubated with control media, control platelets, RGDS treated platelets, anti-CD47 Ab treated platelets, or with CD47^−/− platelets overnight and then LPS stimulated. n=6 replicates from 2 mice. B) Monocytes were incubated with control media, control platelets or RBCs overnight and then LPS treated. n=4 replicates from 2 mice. C) Mice were treated with anti-CD47 Ab or control IgG on d0, d2, and d4. On d5, peripheral monocytes were isolated for qRT-PCR or cultured and stimulated with LPS for 24 hs ex vivo. n=5–6 mice in each group D) Thrombocytopenic mice were transfused with saline, WT platelets or CD47^−/− platelets. 24 hrs later, mice were treated with LPS and plasma IL-6 and TNFα were quantified 4 hrs later. n=4 mice in each group. E) WT or CD47^−/− monocytes were incubated with either WT or CD47^−/− platelets overnight and LPS stimulated after platelets removed. n=6 replicates from 3 mice. F-G) Resting platelets limit macrophage phagocytosis and increase monocyte migration to chemokines. F) BMDMs were treated with platelets overnight and then co-cultured with latex bead-rabbit FITC-IgG for 2 hrs. Phagocytosis was quantified by flow cytometry. n=6 replicates from 2 mice. G) Platelet pre-treated monocytes were seeded in the top of trans-well chamber and CCL2 in the bottom chamber. 24 hrs later, migrated monocytes were stained with crystal violet and quantified under microscope. 20 random fields were assessed per group; n=4 replicates from 2 mice. The representative images were chosen based on their quality and to most accurately reflect the group average across all the available data. Data were represented as mean±SEM. Statistics: One-way ANOVA followed by Tukey’s multiple comparisons test in A, E and F; Kruskal-Wallis followed by Conover-Iman post hoc test with Holm-sidak corrections in B, D and G; Unpaired, 2-tailed Student’s t test in C (circulating monocyte gene expression); Mann-Whitney test in C (LPS treated ex vivo); Scale bar, 200μm.

Figure 5.

Resting platelets durably program monocytes. A) Platelet mediated effects on monocyte/macrophage LPS responses are durable over many days. Monocytes were incubated with platelets overnight and washed to remove platelets. Cells were then LPS stimulated 48 hrs later. IL-6 production was determined 24hs later and for proliferation, cells were stained with CFSE before platelet-co-culture. 24 hs after LPS treatment, proliferation was assessed by CFSE dye dilution using flow cytometry. Similarly, platelets limited RAW cell LPS induced IL-6 up to 6d after platelet removal. n=4 replicates from 3 mice. B-D) Circulating monocytes were isolated on d5 post-DT from control and thrombocytopenic mice for ATAC-Seq. n=3 mice in each group. B) PCA plot showed monocytes from control and thrombocytopenic mice had different chromatin configurations. C) GO analysis on more accessible monocyte DNA regions from thrombocytopenic mice were related to inflammatory and bacteria responses. D) Venn diagram of the intersection between altered LPS response genes in RNA-Seq and ATAC-Seq from thrombocytopenic mouse monocytes compared to WT monocytes. Heatmap showing normalized accessibility fold change (relative to WT monocyte chromatin accessibility) of those 30 LPS response associated gene regions. Data were represented as mean±SEM. Statistics: Mann-Whitney test in A (LPS stimulated 48h later); 2-way aligned rank transformation (ART) ANOVA with Sidak corrections in A (LPS stimulated 4d or 6d later).

Figure 6.

Resting platelets program monocytes through histone methylation. A) Monocytes were treated with inhibitor of HDACs (SAHA), histone demethylase inhibitor (pargyline) or histone methyltransferase inhibitor (MTA) prior to co-culture with platelets overnight and then platelets washed away and LPS stimulated. n=4 replicates from 2 mice. B) Immunoblots of H3K9me3, H3K27me3 and H3K9ac in circulating monocytes from CD47^−/−, thrombocytopenic and WT mice. n=3 mice in each group. C) Immunoblots of H3K9me3, H3K27me3 and H3K9ac in monocytes cultured with control media, WT platelets or CD47^−/− platelets in vitro overnight. n=3 replicates from 3 mice. Data were represented as mean±SEM. Statistics: 2-way aligned rank transformation (ART) ANOVA with Sidak corrections in A; Kruskal-Wallis followed by Conover-Iman post hoc test with Holm-sidak corrections in B and C.

Figure 7.

Platelet mediated metabolic programming leads to monocyte histone methylation in a platelet CD47 dependent manner. A) Glycolytic rate assays were performed on monocytes incubated with control media, WT platelets, CD47 blocking Ab treated, or CD47^−/− platelets overnight. n=6 replicates from 6 mice. B) Immunoblots of phosphorylated AKT and mTOR in monocytes following overnight culture with control buffer, WT or CD47^−/− platelets. n=3 replicates from 3 mice. C) Monocytes were treated with mTOR or AKT inhibitors prior to platelet co-culture, platelets washed away and monocytes were treated with LPS. n=3 replicates from 2 mice. D) Immunoblots of H3K9me3, H3K27me3 and H3K9ac in monocytes treated with inhibitors of AKT (MK2206) or mTOR (Rapamycin) prior to co-culture with platelets in vitro. n=3 replicates from 4 mice. Data were represented as mean±SEM. Statistics: One-way ANOVA followed by Tukey’s multiple comparisons test in A; Kruskal-Wallis followed by Conover-Iman post hoc test with Holm-sidak corrections in B and C; 2-way aligned rank transformation (ART) ANOVA with Sidak corrections in D.

Figure 8.

Platelet mediated monocyte glycolysis is essential for monocyte immune programming. A) Immunoblots of H3K9me3, H3K27me3 and H3K9ac in monocytes cultured with platelets under control media or glucose-free media. n=3 replicates from 2 mice. B-C) Monocytes were treated with B) 2-DG prior to platelet co-culture or C) co-culture performed in glucose-free media for 24 hrs and then LPS treated. n=3 replicates from 2 mice. Data were represented as mean±SEM. Statistics: 2-way aligned rank transformation (ART) ANOVA with Sidak corrections in A, B and C.