Intermittent intense ultraviolet (UV) exposure represents an important aetiological factor in the development of malignant melanoma. The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is now firmly established, but how the microenvironmental effects of UV radiation influence melanoma pathogenesis is not fully understood. Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression, independent of its tumour-initiating effects. UV irradiation enhanced the expansion of tumour cells along abluminal blood vessel surfaces and increased the number of lung metastases. This effect depended on the recruitment and activation of neutrophils, initiated by the release of high mobility group box 1 (HMGB1) from UV-damaged epidermal keratinocytes and driven by Toll-like receptor 4 (TLR4). The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system, but also show that the resulting inflammatory response catalyses reciprocal melanoma-endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists. Angiotropism represents a hitherto underappreciated mechanism of metastasis that also increases the likelihood of intravasation and haematogenous dissemination. Consistent with our findings, ulcerated primary human melanomas with abundant neutrophils and reactive angiogenesis frequently show angiotropism and a high risk for metastases. Our work indicates that targeting the inflammation-induced phenotypic plasticity of melanoma cells and their association with endothelial cells represent rational strategies to specifically interfere with metastatic progression.

G proteins are an important class of regulatory switches in all living systems. They are activated by guanine nucleotide exchange factors (GEFs), which facilitate the exchange of GDP for GTP. This activity makes GEFs attractive targets for modulating disease-relevant G-protein-controlled signalling networks. GEF inhibitors are therefore of interest as tools for elucidating the function of these proteins and for therapeutic intervention; however, only one small molecule GEF inhibitor, brefeldin A (BFA), is currently available. Here we used an aptamer displacement screen to identify SecinH3, a small molecule antagonist of cytohesins. The cytohesins are a class of BFA-resistant small GEFs for ADP-ribosylation factors (ARFs), which regulate cytoskeletal organization, integrin activation or integrin signalling. The application of SecinH3 in human liver cells showed that insulin-receptor-complex-associated cytohesins are required for insulin signalling. SecinH3-treated mice show increased expression of gluconeogenic genes, reduced expression of glycolytic, fatty acid and ketone body metabolism genes in the liver, reduced liver glycogen stores, and a compensatory increase in plasma insulin. Thus, cytohesin inhibition results in hepatic insulin resistance. Because insulin resistance is among the earliest pathological changes in type 2 diabetes, our results show the potential of chemical biology for dissecting the molecular pathogenesis of this disease.

Summary Adaptive cellular immunity is initiated by antigen-specific interactions between T lymphocytes and dendritic cells (DC). Plasmacytoid DC (pDC) support antiviral immunity by linking innate and adaptive immune responses. Here we examined pDC spatiotemporal dynamics during viral infection to uncover when, where and how they exert their functions. We found that pDC accumulated at sites of CD8+ T cell antigen-driven activation in a CCR5-dependent fashion. Furthermore, activated CD8+ T cells orchestrated the local recruitment of lymph node resident XCR1 chemokine receptor-expressing DC via secretion of the XCL1 chemokine. Functionally, this CD8+ T cell mediated reorganization of the local DC network allowed for the interaction and cooperation of pDC and XCR1+ DC, thereby optimizing XCR1+ DC maturation and cross-presentation. These data support a model in which CD8+ T cells upon activation create their own optimal priming microenvironment by recruiting additional DC subsets to the site of initial antigen recognition.

Cross-priming allows dendritic cells (DCs) to induce cytotoxic T cell (CTL) responses to extracellular antigens. DCs require cognate 'licensing' for cross-priming, classically by helper T cells. Here we demonstrate an alternative mechanism for cognate licensing by natural killer T (NKT) cells recognizing microbial or synthetic glycolipid antigens. Such licensing caused cross-priming CD8alpha(+) DCs to produce the chemokine CCL17, which attracted naive CTLs expressing the chemokine receptor CCR4. In contrast, DCs licensed by helper T cells recruited CTLs using CCR5 ligands. Thus, depending on the type of antigen they encounter, DCs can be licensed for cross-priming by NKT cells or helper T cells and use at least two independent chemokine pathways to attract naive CTLs. Because these chemokines acted synergistically, this can potentially be exploited to improve vaccinations.

Neutrophils are potent immune effectors against bacterial infections. Macrophages are important in infections as effectors and regulators, but their exact roles, phenotypic characterization and their relation to neutrophils is incompletely understood. Here we report in a model of bacterial urinary tract infection, one of the most prevalent bacterial infections that tissue-resident Ly6C− macrophages recruited circulating neutrophils and inflammatory Ly6C+ macrophages through chemokines. Neutrophils were primarily recruited through ligands of the chemokine receptor CXCR2, in particular by CXCL1 and less by macrophage migration inhibitory factor (MIF), but not through CXCL5 and CXCL2. Neutrophils, but not Ly6C+ macrophages, cleared the bacteria by phagocytosis. Ly6C+ macrophages instead performed a regulatory function: in response to the infection, they produced the cytokine tumor necrosis factor (TNF), which in turn caused the resident macrophages to secrete CXCL2. This chemokine induced the secretion of matrix metalloproteinase-9 (MMP-9) in neutrophils and allowed these cells to degrade the uroepithelial basement membrane, in order to enter the uroepithelium, the mucosal interface from where the bacteria invade the bladder. Thus, the phagocyte response against bacteria is a highly coordinated event, in which Ly6C− macrophages act as sentinels and Ly6C+ macrophages as innate helper cells. In analogy with T helper cells (Th), we propose to name these helper macrophages (Ph) as they provide a second signal on whether to unleash the principal effector phagocytes, the neutrophils. This cellular triage may prevent ‘false-positive’ immune responses. The role of TNF as innate ‘licensing’ factor contributes to its central role in antibacterial immunity.