Heterocomplexes between the Atypical Chemokine MIF and the CXC-Motif Chemokine CXCL4L1 Regulate Inflammation and Thrombus Formation

Abstract

AbstractTo fulfil its orchestrating function in immune cell trafficking, a network of chemokines and receptors capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within the network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to the chemo-kine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine-chemokine interactions extend to ACKs and that MIF may form heterocomplexes with classical chemokines. We tested this hypothesis by an unbiased chemokine protein array. The platelet chemokine CXCL4L1, but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12, was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation abrogated MIF-elicited T-cell chemotaxis as assessed in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ. Of note, CXCL4L1 blocked the binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. As MIF and CXCL4L1 are platelet products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and clinical human thrombus sections. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction, adding to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures to modulate inflammation and thrombosis.