Heterocomplexes between the atypical chemokine MIF and the CXC-motif chemokine CXCL4L1 regulate inflammation and thrombus formation

Abstract

To fulfil its orchestration of immune cell trafficking, a network of chemokines and receptors developed that capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within this 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 chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine–chemokine interactions extend to ACKs and that MIF forms heterocomplexes with classical chemokines. We tested this hypothesis by using an unbiased chemokine protein array. 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 was observed to inhibit MIF-elicited T-cell chemotaxis as assessed by transwell migration assay and in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ, as well as MIF-mediated monocyte adhesion on aortic endothelial cell monolayers under flow stress conditions. Of note, CXCL4L1 blocked 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. Because MIF and CXCL4L1 are platelet-derived products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and in clinical human thrombus sections obtained from peripheral artery disease (PAD) in patients undergoing thrombectomy. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction that adds 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 that can be exploited to modulate inflammation and thrombosis.

Impact of the MIF -173G/C variant on cardiovascular disease risk: a meta-analysis of 9,047 participants

“…In addition to its direct effects on inflammation and plaque stability, MIF interacts intricately with CXCL4L1, leading to the formation of prothrombotic and proinflammatory MIF-CXCL4L1 heterocomplexes ( 47 , 48 ). These heterocomplexes have been implicated in promoting endothelial dysfunction, thrombosis, and exacerbation of inflammatory responses within the vascular environment ( 49 ). The presence of the -173 polymorphism in the MIF gene may modulate the formation or activity of these heterocomplexes, potentially influencing the progression and severity of CAD.…”

Molecular Events in Immune Responses to Sublingual Influenza Vaccine with Hemagglutinin Antigen and Poly(I:C) Adjuvant in Nonhuman Primates, Cynomolgus Macaques

“…The upregulation of PF4V1 observed in the present study using the influenza HA antigen and the same adjuvant suggests that the PF4V1 induction is not antigen-specific but related to the inflammatory effect of the Poly(I:C) adjuvant. Brandhofer et al reported that PF4V1 forms a heterocomplex with an atypical chemokine macrophage migration inhibitory factor (MIF) and that the PF4V1-MIF complex does not have chemotactic activity to T cells and monocytes [49]. The upregulation of PF4V1 may have a suppressive aspect for inflammation caused by adjuvants.…”

Heterologous Interactions with Galectins and Chemokines and Their Functional Consequences

“…Moreover, the formation of the CXCL4/CCL5 heterodimer was validated experimentally both in vitro and in vivo [ 5 , 6 ]. More recently, Brandhofer et al [ 55 ] demonstrated that heterocomplexes also form between atypical chemokine MIF (macrophage migration inhibitory factor) and CXCL4L1 and regulate inflammation and thrombus formation in patients with peripheral artery disease.…”