TY - JOUR T1 - Heterocomplexes between the Atypical Chemokine MIF and the CXC-Motif Chemokine CXCL4L1 Regulate Inflammation and Thrombus Formation JF - bioRxiv DO - 10.1101/2021.11.26.470090 SP - 2021.11.26.470090 AU - Markus Brandhofer AU - Adrian Hoffmann AU - Xavier Blanchet AU - Elena Siminkovitch AU - Anne-Katrin Rohlfing AU - Omar El Bounkari AU - Jeremy A. Nestele AU - Alexander Bild AU - Christos Kontos AU - Kathleen Hille AU - Vanessa Rohde AU - Adrian Fröhlich AU - Jona Golemi AU - Ozgun Gokce AU - Christine Krammer AU - Patrick Scheiermann AU - Nikolaos Tsilimparis AU - Wolfgang E. Kempf AU - Lars Maegdefessel AU - Remco T.A. Megens AU - Hans Ippel AU - Rory R. Koenen AU - Kevin H. Mayo AU - Meinrad Gawaz AU - Aphrodite Kapurniotu AU - Christian Weber AU - Philipp von Hundels-hausen AU - Jürgen Bernhagen Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/02/24/2021.11.26.470090.abstract N2 - To fulfil their orchestrating function in immune cell trafficking in homeostasis and disease, a network of 49 chemokines and 23 receptors capitalizes on features of specificity, redundancy, and functional selectivity such as biased agonism. The discovery of the chemokine interactome, i.e. heteromeric chemokine-chemokine interactions, even across CC- and CXC-class borders, has further expanded the complexity within the network. Moreover, some inflammatory mediators, which are not structurally linked to classical CC-, CXC-, CX3C-, or C-chemokines, can bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified the cytokine macrophage migration inhibitory factor (MIF) as an ACK that binds to the chemokine 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, applying an unbiased chemokine protein binding 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, which also established high-affinity binding (KD≍100-150 nM). The binding interface was predicted by peptide array-based mapping and molecular docking. We next determined whether heterocomplex formation modulates inflammatory and atherogenic activities of MIF. MIF-elicited T-cell chemotaxis as assessed in a 3D-matrix-based live cell-imaging set-up was abrogated, when cells were co-incubated with MIF and CXCL4L1. Heterocomplexation also blocked MIF-triggered migration of Egfp+ microglia in cortical cultures in situ. Of note, CXCL4L1 blocked the binding of Alexa-MIF to a soluble ectodomain mimic of CXCR4 and co-incubation with CXCL4L1 attenuated MIF-triggered dynamic mass redistribution in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. As MIF and CXCL4L1 are abundant 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, heterocomplex formation inhibited MIF- stimulated thrombus formation under flow and skewed the morphology of adhering platelets from a large to a small lamellipodia phenotype. Together, 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 promising target structures to modulate inflammation and thrombosis.Competing Interest StatementJ.B., C.W., and A.K. are inventors on patent applications related to anti-MIF and anti-chemokine strategies in inflammatory and cardiovascular diseases. The other authors have no competing interests. ER -