Chemokines and adhesion molecules upregulated in lymphatic endothelial cells (LECs) during tissue inflammation are thought to enhance dendritic cell (DC) migration to draining lymph nodes, but the in vivo control of this process is not well understood. We performed a transcriptional profiling analysis of LECs isolated from murine skin and found that inflammation induced by a contact hypersensitivity (
tributed to the design of experiments, data analysis, and interpretation of results. PB provided transgenic mice. DMM contributed to the design of experiments, data analysis, interpretation of results, and writing of the manuscript.
IntroductionPeripheral lymph nodes (PLNs) are strategically positioned at the junctions of the blood and lymphatic vascular systems to efficiently contain and present microbes or derived peptides to passing lymphocytes for a rapid initiation of adaptive immune responses. [1][2][3][4] The PLN parenchyma is filled with mobile lymphocytes organized in separate T-and B-cell microenvironments in the paracortex and follicles, respectively. PLNs contain antigen-presenting cells (APCs), in particular dendritic cells (DCs) in the T-cell area, which are embedded in a network of fibroblastic stromal cells (FRCs) and high endothelial venules (HEVs), the main port of entry for circulating lymphocytes. [4][5][6][7] When live microbes or microbial products invade a host, draining PLNs undergo substantial morphologic changes to allow for increased recruitment of naive cells. Macroscopically, this is manifested in PLN swelling and an increase in the lymph and blood vasculature. [8][9][10][11][12][13][14][15] In parallel, immunohistologic analysis of the distribution of APC, lymphocyte subsets, and stromal markers points to a substantial microenvironmental reorganization in draining PLNs. 12,16 These changes are in part caused by a temporary shutdown of lymphocyte egress, recruitment of new lymphocytes and activated DCs, as well as retention and clonal expansion of antigen-specific lymphocytes. 4,17 Expression levels of homeostatic chemokines, such as CCL19 and CCL21, decrease in inflamed lymphoid tissue, either through interferon-␥-dependent mechanisms or through destruction of the stromal network producing these chemotactic factors. As an example, infection with the model pathogen lymphocytic choriomeningitis virus (LCMV) results in 10-fold-reduced CCL19 and CCL21 mRNA levels, precipitating the loss of lymphoid organization. 17,18 The inflammation-induced PLN expansion is an almostunparalleled process of tissue remodeling regarding its rapidity and the extent of size increase in adult organisms. The increase in blood vasculature implies a role for the proangiogenic factor vascular endothelial growth factor A (VEGF-A) during PLN expansion. Accordingly, in delayed-type hypersensitivity responses or adjuvansmediated PLN remodeling, VEGF-A regulates lymph and blood vessel angiogenesis in B cell-dependent and -independent manners. 12,14,15 Activated DCs also contribute to HEV expansion in a VEGF-A-dependent manner. 14 FRCs contribute to PLN growth through secretion of VEGF-A after stimulation of their lymphotoxin  receptor (LTR). 19 LTR is furthermore expressed by HEV and lymphatic vessels and is involved in regulation of the HEV phenotype in steady state and inflammation. 13,20 The precise contribution of these morphogenic cells and factors to PLN growth is likely to vary according to the inflammatory stimuli used. For example, because LCMV infection leads to a down-regulation of FRC and DC numbers in draining PLNs, 17 these cells are unlikely to decisively contribute to global lymphoid Submitted October 19, 2009; accepted Janua...
To induce adaptive immunity, dendritic cells (DCs) migrate through afferent lymphatic vessels (LVs) to draining lymph nodes (dLNs). This process occurs in several consecutive steps. Upon entry into lymphatic capillaries, DCs first actively crawl into downstream collecting vessels. From there, they are next passively and rapidly transported to the dLN by lymph flow. Here, we describe a role for the chemokine CCL21 in intralymphatic DC crawling. Performing time-lapse imaging in murine skin, we found that blockade of CCL21-but not the absence of lymph flow-completely abolished DC migration from capillaries toward collecting vessels and reduced the ability of intralymphatic DCs to emigrate from skin. Moreover, we found that in vitro low laminar flow established a CCL21 gradient along lymphatic endothelial monolayers, thereby inducing downstream-directed DC migration. These findings reveal a role for intralymphatic CCL21 in promoting DC trafficking to dLNs, through the formation of a flow-induced gradient.
Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing redfluorescent vasculature and yellowfluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steadystate and inflammation. (Blood. 2012; 120(11):2249-2258) IntroductionDendritic cells (DCs) are important in the initiation of adaptive immune responses. In peripheral tissues, such as the skin, DCs take up antigen, mature, and migrate via lymphatic vessels (LVs) to draining lymph nodes (dLNs), where they present antigen to resting T cells. Although this migration pattern has been known for more than 20 years, 1 DC migration into LVs is only now starting to be unraveled at the cellular level using live imaging technologies. [2][3][4] Before transmigrating across the lymphatic endothelium, DCs first need to squeeze through preformed, narrow pores present in the lymphatic basement membrane (BM). 3 DC entry into LVs is thought to occur at the level of primary lymphatic capillaries, which feature discontinuous cell junctions with "button"-like accumulations of cell adhesion molecules. 5 At the sites of such buttons, lymphatic endothelial cells (LECs) partially overlap and generate open flaps, through which leukocytes enter into lymphatics. 3,5 The most prominent mediator of DC migration into afferent lymphatics is CCL21, a chemokine constitutively expressed in LVs. 6,7 More recently, also other LEC-expressed molecules that mediate DC migration via LVs to dLNs have been identified. [8][9][10][11] Notably, ICAM-1 and VCAM-1, which are up-regulated in LVs during inflammation, 8,12 have been implicated in this process. 8 Intriguingly, experiments performed with pan-integrin knockout DCs revealed that DC migration to dLNs in steady-state was integrin-independent. 2 This ...
Oncolytic viruses pose many questions in their use in cancer therapy. In this study, we assessed the potential of mpJX-594 (mouse-prototype JX-594), a replication-competent vaccinia virus administered by intravenous injection, to target the tumor vasculature, produce immune activation and tumor cell killing more widespread than the infection, and suppress invasion and metastasis. These actions were examined in RIP-Tag2 transgenic mice with pancreatic neuroendocrine tumors that developed spontaneously and progressed as in humans. mpJX-594 initially infected tumor vascular endothelial cells, leading to vascular pruning and prolonged leakage in tumors but not in normal organs; parallel effects were observed in U87 gliomas. Viral infection spread to tumor cells, where tumor cell killing was much more widespread than the infection. Widespread tumor cell killing at 5 days was prevented by depletion of CD8 T lymphocytes and did not require GM-CSF, as mpJX-594 variants that expressed human, mouse, or no GM-CSF produced equivalent amounts of killing. The antivascular, antitumor, and antimetastatic effects of mpJX-594 were amplified by concurrent or sequential administration of sunitinib, a multitargeted receptor tyrosine kinase inhibitor. These effects were not mimicked by selective inhibition of VEGFR2 despite equivalent vascular pruning, but were accompanied by suppression of regulatory T cells and greater influx of activated CD8 T cells. Together, our results showed that mpJX-594 targets tumor blood vessels, spreads secondarily to tumor cells, and produces widespread CD8 T-cell-dependent tumor cell killing in primary tumors and metastases, and that these effects can be amplified by coadministration of sunitinib. These findings reveal multiple unrecognized features of the antitumor properties of oncolytic vaccinia viruses, all of which can be amplified by the multitargeted kinase inhibitor sunitinib. .
Classical type 1 dendritic cells (cDC1s) are required for anti-viral and anti-tumor immunity, which necessitates an understanding of their development. Development of the cDC1 progenitor requires an E protein–dependent enhancer located 41 kilobases downstream of the transcription start site of the transcription factor IRF8 (+41 kb Irf8 enhancer) but its maturation instead requires the BATF3-dependent +32 kb Irf8 enhancer. To understand this switch, we performed single-cell RNA sequencing of the common dendritic cell progenitor (CDP) and identified a cluster of cells that expressed transcription factors that influence cDC1 development, such as Nfil3, Id2 , and Zeb2 . Genetic epistasis among these factors revealed that Nfil3 expression is required for the transition from Zeb2 hi and Id2 lo CDPs to Zeb2 lo and Id2 hi CDPs, which represent the earliest committed cDC1 progenitors. This genetic circuit blocks E protein activity to exclude plasmacytoid DC potential and explains the switch in Irf8 enhancer usage during cDC1 development.
Dendritic cells (DCs) are thought to form a dendritic network across barrier surfaces and throughout organs, including the kidney, to perform an important sentinel function. However, previous studies of DC function used markers, such as CD11c or CX3CR1, that are not unique to DCs. Here, we evaluated the role of DCs in renal inflammation using a CD11c reporter mouse line and two mouse lines with DC-specific reporters, Zbtb46-GFP and Snx22-GFP. Multiphoton microscopy of kidney sections confirmed that most of the dendritically shaped CD11c + cells forming a network throughout the renal interstitium expressed macrophage-specific markers. In contrast, DCs marked by Zbtb46-GFP or Snx22-GFP were less abundant, concentrated around blood vessels, and round in shape. We confirmed this pattern of localization using imaging mass cytometry. Motility measurements showed that resident macrophages were sessile, whereas DCs were motile before and after inflammation. Although uninflamed glomeruli rarely contained DCs, injury with nephrotoxic antibodies resulted in accumulation of ZBTB46 + cells in the periglomerular region. ZBTB46 identifies all classic DCs, which can be categorized into two functional subsets that express either CD103 or CD11b. Depletion of ZBTB46 + cells attenuated the antibody-induced kidney injury, whereas deficiency of the CD103 + subset accelerated injury through a mechanism that involved increased neutrophil infiltration. RNA sequencing 7 days after nephrotoxic antibody injection showed that CD11b + DCs expressed the neutrophil-attracting cytokine CXCL2, whereas CD103 + DCs expressed high levels of several anti-inflammatory genes. These results provide new insights into the distinct functions of the two major DC subsets in glomerular inflammation.
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