Platelet-derived β2M regulates monocyte inflammatory responses

Abstract

β-2 Microglobulin (β2M) is a molecular chaperone for the major histocompatibility class I (MHC I) complex, hemochromatosis factor protein (HFE), and the neonatal Fc receptor (FcRn), but β2M may also have less understood chaperone-independent functions. Elevated plasma β2M has a direct role in neurocognitive decline and is a risk factor for adverse cardiovascular events. β2M mRNA is present in platelets at very high levels, and β2M is part of the activated platelet releasate. In addition to their more well-studied thrombotic functions, platelets are important immune regulatory cells that release inflammatory molecules and contribute to leukocyte trafficking, activation, and differentiation. We have now found that platelet-derived β2M is a mediator of monocyte proinflammatory differentiation through noncanonical TGFβ receptor signaling. Circulating monocytes from mice lacking β2M only in platelets (Plt-β2M-/-) had a more proreparative monocyte phenotype, in part dependent on increased platelet-derived TGFβ signaling in the absence of β2M. Using a mouse myocardial infarction (MI) model, Plt-β2M-/- mice had limited post-MI proinflammatory monocyte responses and, instead, demonstrated early proreparative monocyte differentiation, profibrotic myofibroblast responses, and a rapid decline in heart function compared with WT mice. These data demonstrate a potentially novel chaperone-independent, monocyte phenotype-regulatory function for platelet β2M and that platelet-derived 2M and TGFβ have opposing roles in monocyte differentiation that may be important in tissue injury responses.

Keywords: Platelets; Vascular Biology.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Platelets are a major source of plasma β2M.

(A) Activated platelets release β2M. Mouse platelets were isolated and treated with control buffer, ADP (10 μM), or thrombin (1 U/ml). β2M release was measured by ELISA (n = 4, *P < 0.05, **P < 0.01, 1-way ANOVA with Bonferroni correction). (B) Platelets, but not WBC, from Plt-β2M–/– mice lack MHC I surface expression. Anti–MHC I or control IgG was incubated with circulating platelets or CD45+ cells (insert) from WT and Plt-β2M–/–. MHC I was quantified by flow cytometry (n = 3, **P < 0.01, 1-way ANOVA with Bonferroni correction). (C) Platelets from WT and Plt-β2M–/– mice had similar activation and aggregation. Washed WT and Plt-β2M–/– platelets were stimulated with ADP, and surface P-selectin was measured. Platelets were also labeled with APC or PE antibodies, thrombin stimulated and platelet aggregates determined as double-positive cells by flow cytometry (n = 4 for both panels, 1-way ANOVA with Bonferroni correction). (D) Platelets are a major source of plasma β2M. Concentration of plasma β2M in 10-week-old WT and Plt-β2M–/– mice was determined by ELISA (n = 5, WT; n = 6, Plt-β2M–/–; *P < 0.05 vs. WT, unpaired 2-tailed t test with Welch’s correction).

Figure 2. β2M induced monocyte inflammatory responses.

(A) THP-1 cells were treated with β2M, and IL-8 release was determined by ELISA. β2M induced inflammatory molecule production in a time- and dose-dependent manner (n = 4, *P < 0.05, **P < 0.01 vs. 0, 1-way ANOVA with Bonferroni correction). (B and C) β2M induced a primary mouse monocyte proinflammatory phenotype. Mouse BM monocytes were isolated and treated with control PBS or β2M (5 μg/ml). (B) Forty-eight hours later, Ly6C surface expression was measured by flow cytometry (n = 9, control; n = 8, β2M; **P < 0.01, unpaired 2-tailed t test with Welch’s correction). (C) KC and IL-6 release determined by ELISA (n = 9, control; n = 8, β2M; **P < 0.01, unpaired 2-tailed t test with Welch’s correction). (D) β2M induced a human monocyte inflammatory phenotype. Human peripheral blood monocytes were isolated and treated with control PBS or β2M (10 μg/ml). CD16 surface expression was measured by flow cytometry (right panel, n = 4, *P < 0.05, unpaired 2-tailed t test with Welch’s correction) and IL-8 release by ELISA (left panel, n = 4 control, n = 7 β2M, *P < 0.05, unpaired 2-tailed t test with Welch’s correction). All graphs represent experiments that were repeated at least twice.

Figure 3. Platelets induce a monocyte proinflammatory phenotype in a β2M-dependent manner.

(A–B) Mouse monocytes were incubated with control buffer, the releasate from WT platelets ± anti-β2M antibody (A), or Plt-β2M–/– mouse platelet releasate (B) (10:1 platelet/monocyte ratio). Control WT platelet releasate induced Ly6Chi monocytes more than anti-β2M antibody (n = 4 control, n = 6 releasate ± anti-β2M Ab, *P < 0.05 vs. control, 1-way ANOVA with Bonferroni correction) (A) or Plt-β2M–/– mouse platelet releasate (B) (n = 4, control; n = 3, WT; Plt-β2M–/–; *P < 0.05 vs. control, 1-way ANOVA with Bonferroni correction). (C) Releasate from β2M–/– platelets did not induce mouse monocyte KC, but instead induced IL-10 release (both panels, n = 4; *P < 0.01 vs. control, 1-way ANOVA with Bonferroni correction). (D) Plt-β2M–/– releasate–induced IL-10 is TGFβ dependent. Anti-TGFβ antibody (10 μg/ml) blocked Plt-β2M–/– releasate–induced IL-10 production (n = 4; *P < 0.05, 1-way ANOVA with Bonferroni correction). All graphs and tables are representative from experiments repeated at least twice.

Figure 4. β2M proinflammatory phenotype signaling is through noncanonical TGFβ receptor signaling.

(A) Inhibition of TGFβR signaling ameliorated β2M-induced monocyte activation. Mouse monocytes were incubated with control buffer, β2M, or β2M and a TGFβR1 kinase inhibitor (SB431542). KC production was determined 48 hours later (n = 4; **P < 0.01, 1-way ANOVA with Bonferroni correction). (B) β2M binds to TGFβR1 and TGFβR2. Fc-TGFβR1 or Fc-TGFβR2 were immobilized on a sensor chip, and β2M, TGFβ1, or TGFβ3 binding was determined by SPR. (C and D) WT and TGFβR2–/– monocytes were incubated with control buffer or β2M. (C) β2M induced WT, but not TGFβR–/– monocyte, Ly6Chi phenotype. (D) Quantification of C (n = 4; *P < 0.05, 1-way ANOVA with Bonferroni correction).

Figure 5. β2M signals through a noncanonical TGFβ receptor signaling mechanism.

(A) Inhibition of TAK1 (7-ox, 1 μM) blocked β2M-induced THP-1 IL-8 but had no effect on β2M-induced SMAD3 activation (P-SMAD3). THP-1 cells were treated with TAK1 inhibitor or control buffer prior to β2M. IL-8 was determined by ELISA (n = 4; **P < 0.01 vs. control, 1-way ANOVA with Bonferroni correction), and P-SMAD3 and total SMAD3 were determined by immunoblot (representative image quantified in Supplemental Figure 12 and 13). (B) JNK inhibitor (SP600125, 10 μM) greatly attenuated THP-1 IL-8 production, but p38 inhibitor (SB202190, 10 μM) only partially attenuated IL-8 (both panels, n = 4, **P < 0.01 vs. control, 1-way ANOVA with Bonferroni correction). (C) β2M-induced WT but not TGFβR2–/– monocyte p-JNK. WT and TGFβR2–/– monocytes were incubated with control buffer or β2M, and intracellular p-JNK was determined by flow cytometry (n = 4, *P < 0.05, unpaired 2-tailed t test with Welch’s correction). (D and E) Blocking β2M oligomer formation with NEM inhibited β2M induced monocyte inflammatory phenotype. β2M was pretreated with buffer or NEM and then added to mouse monocytes for 48 hours. (D) NEM reduced β2M oligomer formation (nonreducing gel), and (E) Ly6Chi monocytes (n = 4, *P < 0.05, 1-way ANOVA with Bonferroni correction).

Figure 6. Platelet β2M regulates circulating monocyte differentiation.

(A) Circulating monocytes were isolated from WT and Plt-β2M–/– mice, and proinflammatory and proreparative monocyte gene markers were quantified by qPCR. Plt-β2M–/– mouse monocytes had proreparative monocyte gene expression (all panels, n = 3; *P < 0.05, unpaired 2-tailed t test with Welch’s correction). (B and C) Monocytes from Plt-β2M–/– mice differentiate to proreparative, fibroblast activating, macrophages in vitro. Peripheral blood monocytes from WT and Plt-β2M–/– mice were coincubated with cardiac fibroblasts for 72 hours and macrophage differentiation (B) and fibroblast activation (C) were determined by qPCR, and IL-10 secretion determined by ELISA. Plt-β2M–/––derived monocytes had increased proreparative differentiation and IL-10 secretion and induced more fibroblast activation compared with WT mouse monocytes (B and C, n = 4; *P < 0.05, **P < 0.01 vs. WT, unpaired 2-tailed t test with Welch’s correction).

Figure 7. Platelet-derived β2M mediates monocyte inflammatory responses to myocardial infarction.

(A) Plasma β2M is elevated in humans after MI. Plasma was isolated from healthy subjects and confirmed MI patients. β2M was measured by ELISA (n = 22, control; n = 55, MI; *P < 0.05 vs. control, unpaired 2-tailed t test with Welch’s correction). (B) WT, but not Plt-β2M–/–, mice had increased post-MI plasma β2M. β2M measured before and 1 day after MI by ELISA (n = 5; *P < 0.05 vs. Plt-β2M–/–, 1-way ANOVA with Bonferroni correction). (C) WT mice had increased circulating Ly6Chi monocytes after MI, but Plt-β2M–/– mice had no change from day 0 (n = 3, WT at 0 and 2 days, and Plt-β2M–/– at 0, 2, and 3 days; n = 4, WT at 3 and 18 days, and Plt-β2M–/– at 18 days; n = 5, Plt-β2M–/– at 7 days; n = 6, WT at 7 days; *P < 0.05, **P < 0.01, 1-way ANOVA with Bonferroni correction). (D) Plt-β2M–/– mice had a rapid post-MI decline in heart function compared with WT mice (n = 5, *P < 0.05 vs. Plt-β2M–/–, unpaired 2-tailed t test with Welch’s correction). (E) WT and Plt-β2M–/– mice had similar post-MI monocyte lineage infiltrates (CD68+), but monocytes are associated with areas of ECM deposition (trichrome) in Plt-β2M–/– mice. Representative images 20× images, from day 15 after MI. (F) Fibrosis quantification (n = 3, control; n = 4, day 15 after MI; *P < 0.05, 1-way ANOVA with Bonferroni correction).

Figure 8. Plt-β2M–/– mice have an early proreparative skewed monocyte response to MI.

(A) Plasma inflammatory protein array. WT mice had a day 3 post-MI increase in proinflammatory plasma proteins, whereas Plt-β2M–/– mice had a greater increase in proreparative plasma proteins. (B) Circulating monocytes were isolated from WT and Plt-β2M–/– mice before and 3 days after MI, and qPCR was performed for Cxcl1 and Il10. Monocytes from Plt-β2M–/– mice had less Cxcl1 and increased Il10 expression after MI compared with WT mice (n = 4, **P < 0.01, 1-way ANOVA with Bonferroni correction).

Figure 9. Plt-β2M–/– mice have an early reparative and fibrotic response to MI.

Cardiac mRNA was isolated from control and day-3 post-MI hearts. (A and B) Plt-β2M–/– mouse hearts had increased post-MI markers of proreparative macrophage phenotype (A) (n = 3, normalized to genotype control; **P < 0.01, 1-way ANOVA with Bonferroni correction) and increased fibroblast activation compared with WT mice (C) (n = 3, normalized to genotype control; **P < 0.01, 1-way ANOVA with Bonferroni correction).

Figure 10. Plt-β2M–/– early proreparative responses to MI are at least in part TGFβ dependent.

Plt-β2M–/– mice were treated with anti-TGFβ antibody prior to MI. On day 4 after MI, monocyte and macrophage inflammatory phenotypes and myofibroblast activation were determined. (A–C) Blocking TGFβ in Plt-β2M–/– mice increased circulating monocyte inflammatory cytokine expression (n = 3, **P < 0.01, 1-way ANOVA with Bonferroni correction) (A), decreased cardiac proreparative macrophages (n = 3; *P < 0.05, **P < 0.01, 1-way ANOVA with Bonferroni correction) (B), and decreased myofibroblast activation compared with control Plt-β2M–/– mice (n = 3, **P < 0.01, 1-way ANOVA with Bonferroni correction) (C).