Abstract D-dopachrome tautomerase (D-DT/MIF-2) is an inflammatory cytokine, atypical chemokine (ACK) and member of the macrophage migration-inhibitory factor (MIF) family. While interactions among classical chemokines (CKs) are established, ACK–CK interactions remain underexplored. Here, we screened for MIF-2 binding-partners using a protein array encompassing all CKs and selected ACKs, and validated candidate interactors by surface-plasmon resonance. CCL20/MIP-3α was prioritized based on RNA-sequencing suggesting induction during liver fibrosis. MIF-2/CCL20 complex formation was verified by microscale thermophoresis and interaction interfaces mapped using peptide array and in-silico modeling. The deduced binding-site near the MIF-2 tautomerase pocket was consistent with inhibition of its tautomerase activity by CCL20. We found both proteins abundantly expressed in human liver tissue, with a positive correlation. Pull-down confirmed complex formation and proximity ligation assay demonstrated MIF-2/CCL20 complexes in liver in situ, with higher levels in fibrotic tissue. Functionally, MIF-2/CCL20 complexes suppressed MIF-2–driven CD4⁺ T-cell chemotaxis and fibroblast IL-6 secretion, indicating modulation of immune and stromal responses. This study extends the ACK interactome to MIF-2 and suggests ACK/CK complexes modulate chemokine activities in liver fibrosis.

Background/Objectives: Patients with shock suffer from hyperlactatemia, which can lead to endothelial dysfunction. The use of the adsorber Cytosorb® (CS) is recommended in these patients as it may contribute to higher lactate clearance and hemodynamic stabilization. However, it is unclear whether CS can directly adsorb lactate and can therefore increase lactate clearance. Methods: The Cyto-SOLVE trial included patients undergoing continuous kidney replacement therapy combined with CS application. Patients with a lactate concentration > 2 mmol/L and measurements of lactate pre- and post-adsorber, as well as measurements in the blood 10 min and 1, 3, 6, and 12 h after initiation were selected. Lactate clearance was calculated using the following formula: bloodflow(mL/min) × concentrationpre−post/concentrationpre. A t-test was used with the collected samples. Changes in the lactate concentration and vasopressor requirement were recorded before initiation and at the end of therapy. Results: Sixty-five lactate concentrations were measured pre- and post-CS application, as well as in patients’ blood, in a total of 14 patients (median age of 52 years, 10 males, median SAPS-II 67). There was no significant change in the lactate concentration pre- and post-CS application (mean pre-CS: 6.7 mmol/L, mean post-CS: 6.9 mmol/L, RR: −0.2, 95% confidence interval (CI): −0.4–0.1, p = 0.13, Cohen’s d: 0.90). The mean lactate clearance was −6 mL/min (standard deviation (SD): 21 mL/min), with no correlation with the initial lactate concentration or blood flow. In contrast, the mean lactate clearance measured using the dialyzer was 39 mL/min (SD: 28 mL/min). When comparing values before and after treatment, no significant change was observed in the lactate blood concentrations (mean of 9.0 vs. 8.5 mmol/L), nor in the requirement for vasopressin (median of 1.9 vs. 1.8 IE/h) or norepinephrine (mean of 2.7 vs. 2.6 mg/h). Conclusions: The adsorber CS cannot directly adsorb lactate, unlike kidney replacement therapy. Therefore, it is not suitable for achieving faster extracorporeal lactate elimination. Understanding the adsorption spectrum is of great relevance and should be considered when using CS in clinical practice.

Recent research has uncovered Arabidopsis thaliana proteins that are similar to the human inflammatory cytokine MIF. Plant MIF/D-dopachrome tautomerase (D-DT)-like proteins (MDLs) can interact with human MIF, yet the significance of these findings in living organisms has not been investigated. Given MIF's key role in acute respiratory distress syndrome promoting pulmonary inflammation, pathology, and leukocyte infiltration, here we set out to investigate the interplay between MIF and MDL1, one of three A. thaliana MIF orthologs, in an in vivo mouse model of MIF-induced acute lung injury (ALI). Human MIF and MDL1 were administered to C57BL/6 mice via inhalation, individually or in combination. Inhalation of MIF promoted various parameters of lung injury as evaluated by flow cytometry, immunofluorescence microscopy, RT-qPCR, and ELISA, while MDL1 inhalation alone had no effect. Intriguingly, combined treatment with MIF and MDL1 synergistically enhanced pulmonary infiltration of neutrophils and monocytic cells, accompanied by an upregulation of pro-inflammatory cytokine genes. Thus, the plant-derived MIF ortholog MDL1 potentiates MIF-induced inflammation in ALI. These data support the growing evidence of interactions between plant-derived compounds and human inflammatory mediators and illustrate how they may impact human health.

Next to its classical role in MHC II-mediated antigen presentation, CD74 was identified as a high-affinity receptor for macrophage migration inhibitory factor (MIF), a pleiotropic cytokine and major determinant of various acute and chronic inflammatory conditions, cardiovascular diseases and cancer. Recent evidence suggests that CD74 is expressed in T cells, but the functional relevance of this observation is poorly understood. Here, we characterized the regulation of CD74 expression and that of the MIF chemokine receptors during activation of human CD4+ T cells and studied links to MIF-induced T-cell migration, function, and COVID-19 disease stage. MIF receptor profiling of resting primary human CD4+ T cells via flow cytometry revealed high surface expression of CXCR4, while CD74, CXCR2 and ACKR3/CXCR7 were not measurably expressed. However, CD4+ T cells constitutively expressed CD74 intracellularly, which upon T-cell activation was significantly upregulated, post-translationally modified by chondroitin sulfate and could be detected on the cell surface, as determined by flow cytometry, Western blot, immunohistochemistry, and re-analysis of available RNA-sequencing and proteomic data sets. Applying 3D-matrix-based live cell-imaging and receptor pathway-specific inhibitors, we determined a causal involvement of CD74 and CXCR4 in MIF-induced CD4+ T-cell migration. Mechanistically, proximity ligation assay visualized CD74/CXCR4 heterocomplexes on activated CD4+ T cells, which were significantly diminished after MIF treatment, pointing towards a MIF-mediated internalization process. Lastly, in a cohort of 30 COVID-19 patients, CD74 surface expression was found to be significantly upregulated on CD4+ and CD8+ T cells in patients with severe compared to patients with only mild disease course. Together, our study characterizes the MIF receptor network in the course of T-cell activation and reveals CD74 as a novel functional MIF receptor and MHC II-independent activation marker of primary human CD4+ T cells.

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.

aDepartment of General, Visceral and Transplant Surgery, University Hospital of Munich, Munich, Germany; bMedical Department II, University Hospital, LMU, Munich, Germany; cDZIF Deutsches Zentrum für Infektionsforschung, Partner Site Munich, München, Germany; dDepartment for General, Visceral and Oncological Surgery, St. George Hospital, Leipzig, Germany; eDepartment of Anesthesiology, LMU University Hospital, Campus Großhadern, Munich, Germany; fDepartment of Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany; gDKFZ-Hector Cancer Institute, University Medical Center Mannheim, Heidelberg University, Mannheim, Germany Received: August 4, 2022 Accepted: August 5, 2022 Published online: October 21, 2022

AbstractTo 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 culturesin 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.