A self-generated Toddler gradient guides mesodermal cell migration

The sculpting of germ layers during gastrulation relies on coordinated migration of progenitor cells, yet the cues controlling these long-range directed movements remain largely unknown. While directional migration often relies on a chemokine gradient generated from a localized source, we find that zebrafish ventrolateral mesoderm is guided by the uniformly expressed and secreted protein Toddler/ELABELA/Apela, acting as a self-generated gradient. We show that the Apelin receptor, which is specifically expressed in mesodermal cells, has a dual role during gastrulation, acting as a scavenger receptor to generate a Toddler gradient, and as a chemokine receptor to sense this guidance cue. Thus, we uncover a single receptor-based self-generated gradient as the enigmatic guidance cue that can robustly steer the directional migration of mesoderm through the complex and continuously changing environment of the gastrulating embryo.

POS0417 EXOGENOUS CXCL5 RESTORES ENDOGENOUS BLOOD-TISSUE CHEMOKINE GRADIENT TO IMPROVE SURVIVAL IN MURINE LUPUS

Background:Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease that is potentially fatal. There is an unmet need to improve current therapies. In patients with SLE, we observed that serum CXCL5 levels were significantly lower than healthy control subjects and negatively correlated with disease activity(1-9).Objectives:The aim of this study is to elucidate the effect of supplemental serum CXCL5 in abrogating the pathological processes of SLE.Methods:Ten doses of exogenous CXCL5 (3µg/kg) was administered to 16-week-old Faslpr mice weekly by intravenous injection. Mice were monitored for 10 weeks. Splenic immune profile was measured by flow cytometry. Circulating cytokine and immunoglobulin profile were detected by Luminex technology. Renal function was evaluated by urinary spot albumin creatinine ratio. In situ renal immune cell infiltration and complement 3 deposition were detected by Haematoxylin and Eosin (H&E) and immunohistochemistry staining. The molecular pathways involved were examined by RNA sequencing.Results:In Faslpr mice, intravenous administration of exogenous CXCL5 significantly improved mouse survival with concomitant reduction of autoantibody secretion, proteinuria, complement 3 deposition, neutrophil infiltration and lupus nephritis classes. Through evaluating the changes of immune profile, cytokine profile and molecular pathways, we found that intravenous CXCL5 reduced inflammation via an orchestral effect of regulating neutrophil trafficking and modulating helper T cell-mediated immune response. Pharmacokinetic and real-time Polymerase Chain Reaction studies further demonstrated that this orchestration was triggered by a cascade reaction - restoring vascular under-expressed CXCL5 by an exogenous stimulation, re-establishing the normal serum levels of endogenous CXCL5 and reverting the CXCL5 chemokine gradient between inflamed tissues and blood circulation.Conclusion:Managing the dysregulation of CXCL5 by exogenous supplement may provide a new option for SLE therapy.References:[1]Dufies M, Grytsai O, Ronco C, et al. New CXCR1/CXCR2 inhibitors represent an effective treatment for kidney or head and neck cancers sensitive or refractory to reference treatments. Theranostics. 2019;9(18):5332-5346. doi:10.7150/thno.34681[2]Yildirim K, Colak E, Aktimur R, et al. Clinical Value of CXCL5 for Determining of Colorectal Cancer. Asian Pac J Cancer Prev. Sep 26 2018;19(9):2481-2484. doi:10.22034/apjcp.2018.19.9.2481[3]Wu K, Yu S, Liu Q, Bai X, Zheng X. The clinical significance of CXCL5 in non-small cell lung cancer. Onco Targets Ther. 2017;10:5561-5573. doi:10.2147/ott.s148772[4]Zhao J, Ou B, Han D, et al. Tumor-derived CXCL5 promotes human colorectal cancer metastasis through activation of the ERK/Elk-1/Snail and AKT/GSK3beta/beta-catenin pathways. Mol Cancer. Mar 29 2017;16(1):70. doi:10.1186/s12943-017-0629-4[5]Han KQ, Han H, He XQ, et al. Chemokine CXCL1 may serve as a potential molecular target for hepatocellular carcinoma. Cancer Med. Oct 2016;5(10):2861-2871. doi:10.1002/cam4.843[6]Pappa CA, Tsirakis G, Kanellou P, et al. Monitoring serum levels ELR+ CXC chemokines and the relationship between microvessel density and angiogenic growth factors in multiple myeloma. Cytokine. Dec 2011;56(3):616-20. doi:10.1016/j.cyto.2011.08.034[7]Zhang L, Li H, Ge C, et al. CXCL3 contributes to CD133(+) CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation. Sci Rep. Jun 3 2016;6:27426. doi:10.1038/srep27426[8]Matsubara J, Honda K, Ono M, et al. Reduced plasma level of CXC chemokine ligand 7 in patients with pancreatic cancer. Cancer Epidemiol Biomarkers Prev. Jan 2011;20(1):160-71. doi:10.1158/1055- 9965.epi-10-0397[9]Ma Y, Ren Y, Dai ZJ, Wu CJ, Ji YH, Xu J. IL-6, IL-8 and TNF-alpha levels correlate with disease stage in breast cancer patients. Adv Clin Exp Med. May-Jun 2017;26(3):421-426. doi:10.17219/acem/62120Disclosure of Interests:None declared

Scavenging of soluble and immobilized CCL21 by ACKR4 regulates peripheral dendritic cell emigration

Leukocyte homing driven by the chemokine CCL21 is pivotal for adaptive immunity because it controls dendritic cell (DC) and T cell migration through CCR7. ACKR4 scavenges CCL21 and has been shown to play an essential role in DC trafficking at the steady state and during immune responses to tumors and cutaneous inflammation. However, the mechanism by which ACKR4 regulates peripheral DC migration is unknown, and the extent to which it regulates CCL21 in steady-state skin and lymph nodes (LNs) is contested. Specifically, our previous findings that CCL21 levels are increased in LNs of ACKR4-deficient mice [I. Comerford et al., Blood 116, 4130–4140 (2010)] were refuted [M. H. Ulvmar et al., Nat. Immunol. 15, 623–630 (2014)], and no differences in CCL21 levels in steady-state skin of ACKR4-deficient mice were reported despite compromised CCR7-dependent DC egress in these animals [S. A. Bryce et al., J. Immunol. 196, 3341–3353 (2016)]. Here, we resolve these issues and reveal that two forms of CCL21, full-length immobilized and cleaved soluble CCL21, exist in steady-state barrier tissues, and both are regulated by ACKR4. Without ACKR4, extracellular CCL21 gradients in barrier sites are saturated and nonfunctional, DCs cannot home directly to lymphatic vessels, and excess soluble CCL21 from peripheral tissues pollutes downstream LNs. The results identify the mechanism by which ACKR4 controls DC migration in barrier tissues and reveal a complex mode of CCL21 regulation in vivo, which enhances understanding of functional chemokine gradient formation.

Acute dietary saturated fat intake suppresses human monocyte subset inflammatory and chemokine responses

Background Dietary saturated fat (SF) intake is implicated in increasing cardiovascular risk. Studies suggest SF may incite an inflammatory immune response yet the mechanisms for this remain unclear. Circulating blood monocyte subsets (classical “CD16low” and non-classical/intermediate “CD16high”) are thought to differentially contribute to the macrophage foam cell burden in atherosclerotic plaque, yet they are heterogenous and much is still unknown about their response to dietary lipid. Purpose This dietary intervention study hypothesised that a subset-specific monocyte response occurs following acute SF intake in healthy individuals.Top of Form Methods 10 healthy participants were fed a coconut oil, lauric acid-rich meal. Monocyte subsets were purified from fasting and 4h postprandial blood by fluorescence activated cytometry (FACS) into CD16low and CD16high subsets. Surface marker expression was recorded by FACS, gene expression by qPCR, lipid loading by fluorescence microscopy, cytokine responses by ELISA and mass spectrometry was utilised to trace dietary fat into circulating monocytes. Triglyceride rich lipoprotein (TGRL) was obtained through ultracentrifugation of postprandial blood. TLR agonists LPS (TLR4) and R848 (TLR7/8) were used to replicate infectious stimuli to provoke a cytokine response. Results SF meal intake resulted in a 4h postprandial rise in serum triglycerides (TG). Other than a small rise in HLA-DR in postprandial CD16low monocytes, no changes in CD14, CD16 or CD11c levels were noted. Inflammatory gene expression was downregulated although a rise in lipid handling gene ABCA1 was noted in both subsets. Postprandial monocytes accumulated cytoplasmic neutral lipid droplets (larger and more numerate droplets in CD16low). Mass-spectrometry confirmed entry of dietary lipid into circulating monocytes. Postprandial monocyte functional responses were impaired; lipid-loaded postprandial monocytes were less able to migrate towards a chemokine gradient. Following TLR challenge, the inflammatory response was abrogated in postprandial monocytes, further suppressed by in vitro postprandial TGRL addition. Lipidomic analyses confirmed postprandial monocyte lauric acid accumulation in the form of free fatty acid and TG trilaurin, and 9(S)-, 13(S)-HODE and 17(S)-HDoHE, that are metabolites of docosahexanoic acid (DHA) and possess anti-inflammatory properties. The attached graphical abstract summarises the pertinent findings. Conclusions Acute dietary SF intake of a lauric acid-rich meal results in postprandial monocyte suppression of inflammatory TLR responses, a reduction in the ability to migrate towards a chemokine gradient whilst accumulating cytoplasmic neutral lipid droplets. The immunosuppression may be caused by accumulation of anti-inflammatory metabolites of DHA. Graphical Abstract Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation, UK

Therapeutic Anticoagulation with Argatroban and Heparins Reduces Granulocyte Migration: Possible Impact on ECLS-Therapy?

Introduction. Anticoagulants such as argatroban and heparins (low-molecular-weight and unfractionated) play an immense role in preventing thromboembolic complications in clinical practice. Nevertheless, they can also have a negative effect on the immune system. This study is aimed at investigating the influence of these substances on polymorphonuclear neutrophils (PMNs), whose nonspecific defense mechanisms can promote thrombogenesis. Methods. Blood samples from 30 healthy volunteers were investigated, whereby PMNs were isolated by density gradient centrifugation and incubated with 0.8 μg/mL of argatroban, 1.0 U/mL of low-molecular-weight heparin (LMWH), 1.0 U/mL of unfractionated heparin (UFH), or without drug (control). A collagen-cell mixture was prepared and filled into 3D μ-slide chemotaxis chambers (IBIDI® GmbH, Germany). Stimulation was initiated by using a chemokine gradient of n-formyl-methionine-leucyl-phenylalanine (fMLP), and microscopic observation was conducted for 4.5 hours. The cells’ track length and track straightness, as well as the number of attracted granulocytes, level of ROS (reactive oxygen species) production, and NET (neutrophil extracellular traps) formation, were analyzed and categorized into migration distances and time periods. Results. All three anticoagulants led to significantly reduced PMN track lengths, with UFH having the biggest impact. The number of tracks observed in the UFH group were significantly reduced compared to the control group. Additionally, the UFH group demonstrated a significantly lower track straightness compared to the control. ROS production and NET formation were unaffected. Conclusion. Our data provide evidence that anticoagulants have an inhibitory effect on the extent of PMN migration and chemotactic migration efficiency, thus indicating their potential immune-modulatory and prothrombotic effects.

Integration of urine proteomics and renal single-cell genomics identifies an interferon-gamma response gradient in lupus nephritis

Lupus nephritis, one of the most serious manifestations of systemic lupus erythematosus (SLE), has both a heterogeneous clinical and pathological presentation. For example, proliferative nephritis identifies a more aggressive disease class that requires immunosuppression. However, the current classification system relies on the static appearance of histopathological morphology which does not capture differences in the inflammatory response. Therefore, a biomarker grounded in the disease biology is needed to understand the molecular heterogeneity of lupus nephritis and identify immunologic mechanism and pathways. Here, we analyzed the patterns of 1000 urine protein biomarkers in 30 patients with active lupus nephritis. We found that patients stratify over a chemokine gradient inducible by interferon-gamma. Higher values identified patients with proliferative lupus nephritis. After integrating the urine proteomics with the single-cell transcriptomics of kidney biopsies, it was observed that the urinary chemokines defining the gradient were predominantly produced by infiltrating CD8 T cells, along with natural killer and myeloid cells. The urine chemokine gradient significantly correlated with the number of kidney-infiltrating CD8 cells. These findings suggest that urine proteomics can capture the complex biology of the kidney in lupus nephritis. Patient-specific pathways may be noninvasively tracked in the urine in real time, enabling diagnosis and personalized treatment.

A statistical inference approach to reconstruct intercellular interactions in cell migration experiments

Migration of cells can be characterized by two prototypical types of motion: individual and collective migration. We propose a statistical inference approach designed to detect the presence of cell-cell interactions that give rise to collective behaviors in cell motility experiments. This inference method has been first successfully tested on synthetic motional data and then applied to two experiments. In the first experiment, cells migrate in a wound-healing model: When applied to this experiment, the inference method predicts the existence of cell-cell interactions, correctly mirroring the strong intercellular contacts that are present in the experiment. In the second experiment, dendritic cells migrate in a chemokine gradient. Our inference analysis does not provide evidence for interactions, indicating that cells migrate by sensing independently the chemokine source. According to this prediction, we speculate that mature dendritic cells disregard intercellular signals that could otherwise delay their arrival to lymph vessels.

New insights in chemokine signaling

Chemokine signaling is essential for coordinated cell migration in health and disease to specifically govern cell positioning in space and time. Typically, chemokines signal through heptahelical, G protein-coupled receptors to orchestrate cell migration. Notably, chemokine receptors are highly dynamic structures and signaling efficiency largely depends on the discrete contact with the ligand. Promiscuity of both chemokines and chemokine receptors, combined with biased signaling and allosteric modulation of receptor activation, guarantees a tightly controlled recruitment and positioning of individual cells within the local environment at a given time. Here, we discuss recent insights in understanding chemokine gradient formation by atypical chemokine receptors and how typical chemokine receptors can transmit distinct signals to translate guidance cues into coordinated cell locomotion in space and time.