Environmental Effects on Temperature Stress Resistance in the Tropical Butterfly Bicyclus Anynana

BACKGROUND: The ability to withstand thermal stress is considered to be of crucial importance for individual fitness and species' survival. Thus, organisms need to employ effective mechanisms to ensure survival under stressful thermal conditions, among which phenotypic plasticity is considered a particularly quick and effective one. METHODOLOGY/PRINCIPAL FINDINGS: In a series of experiments we here investigate phenotypic adjustment in temperature stress resistance following environmental manipulations in the butterfly Bicyclus anynana. Cooler compared to warmer acclimation temperatures generally increased cold but decreased heat stress resistance and vice versa. In contrast, short-time hardening responses revealed more complex patterns, with, e.g., cold stress resistance being highest at intermediate hardening temperatures. Adult food stress had a negative effect on heat but not on cold stress resistance. Additionally, larval feeding treatment showed interactive effects with adult feeding for heat but not for cold stress resistance, indicating that nitrogenous larval resources may set an upper limit to performance under heat stress. In contrast to expectations, cold resistance slightly increased during the first eight days of adult life. Light cycle had marginal effects on temperature stress resistance only, with cold resistance tending to be higher during daytime and thus active periods. CONCLUSIONS/SIGNIFICANCE: Our results highlight that temperature-induced plasticity provides an effective tool to quickly and strongly modulate temperature stress resistance, and that such responses are readily reversible. However, resistance traits are not only affected by ambient temperature, but also by, e.g., food availability and age, making their measurement challenging. The latter effects are largely underexplored and deserve more future attention. Owing to their magnitude, plastic responses in thermal tolerance should be incorporated into models trying to forecast effects of global change on extant biodiversity

Intralymphatic CCL21 promotes tissue egress of dendritic cells through afferent lymphatic vessels

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

Locally Triggered Release of the Chemokine CCL21 Promotes Dendritic Cell Transmigration across Lymphatic Endothelia

Trafficking cells frequently transmigrate through epithelial and endothelial monolayers. How monolayers cooperate with the penetrating cells to support their transit is poorly understood. We studied dendritic cell (DC) entry into lymphatic capillaries as a model system for transendothelial migration. We find that the chemokine CCL21, which is the decisive guidance cue for intravasation, mainly localizes in the trans-Golgi network and intracellular vesicles of lymphatic endothelial cells. Upon DC transmigration, these Golgi deposits disperse and CCL21 becomes extracellularly enriched at the sites of endothelial cell-cell junctions. When we reconstitute the transmigration process in vitro, we find that secretion of CCL21-positive vesicles is triggered by a DC contact-induced calcium signal, and selective calcium chelation in lymphatic endothelium attenuates transmigration. Altogether, our data demonstrate a chemokine-mediated feedback between DCs and lymphatic endothelium, which facilitates transendothelial migration.Peer reviewe

Preformed portals facilitate dendritic cell entry into afferent lymphatic vessels

Although both processes occur at similar rates, leukocyte extravasation from the blood circulation is well investigated, whereas intravasation into lymphatic vessels has hardly been studied. In contrast to a common assumption—that intra- and extravasation follow similar molecular principles—we previously showed that lymphatic entry of dendritic cells (DCs) does not require integrin-mediated adhesive interactions. In this study, we demonstrate that DC-entry is also independent of pericellular proteolysis, raising the question of whether lymphatic vessels offer preexisting entry routes. We find that the perilymphatic basement membrane of initial lymphatic vessels is discontinuous and therefore leaves gaps for entering cells. Using a newly developed in situ live cell imaging approach that allows us to dynamically visualize the cells and their extracellular environment, we demonstrate that DCs enter through these discontinuities, which are transiently mechanically dilated by the passaging cells. We further show that penetration of the underlying lymphatic endothelial layer occurs through flap valves lacking continuous intercellular junctions. Together, we demonstrate free cellular communication between interstitium and lymphatic lumen

Microtubules control cellular shape and coherence in amoeboid migrating cells

Cells navigating through complex tissues face a fundamental challenge: while multiple protrusions explore different paths, the cell needs to avoid entanglement. How a cell surveys and then corrects its own shape is poorly understood. Here, we demonstrate that spatially distinct microtubule dynamics regulate amoeboid cell migration by locally promoting the retraction of protrusions. In migrating dendritic cells, local microtubule depolymerization within protrusions remote from the microtubule organizing center triggers actomyosin contractility controlled by RhoA and its exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: (1) impaired cell edge coordination during path finding and (2) defective adhesion resolution. Compromised shape control is particularly hindering in geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. We thus demonstrate that microtubules can act as a proprioceptive device: they sense cell shape and control actomyosin retraction to sustain cellular coherence

Intravital Two-Photon Microscopy of Immune Cell Dynamics in Corneal Lymphatic Vessels

BACKGROUND: The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years. METHODOLOGY/PRINCIPAL FINDINGS: We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1-5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces. CONCLUSIONS: To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions

Regulation of T Cell Priming by Lymphoid Stroma

The priming of immune T cells by their interaction with dendritic cells (DCs) in lymph nodes (LN), one of the early events in productive adaptive immune responses, occurs on a scaffold of lymphoid stromal cells, which have largely been seen as support cells or sources of chemokines and homeostatic growth factors. Here we show that murine fibroblastic reticular cells (FRCs), isolated from LN of B6 mice, play a more direct role in the immune response by sensing and modulating T cell activation through their upregulation of inducible nitric oxide synthase (iNOS) in response to early T cell IFNγ production. Stromal iNOS, which only functions in very close proximity, attenuates responses to inflammatory DC immunization but not to other priming regimens and preferentially affects Th1 cells rather than Th2. The resultant nitric oxide production does not affect T cell-DC coupling or initial calcium signaling, but restricts homotypic T cell clustering, cell cycle progression, and proliferation. Stromal feedback inhibition thus provides basal attenuation of T cell responses, particularly those characterized by strong local inflammatory cues

Target deconvolution of a multikinase inhibitor with antimetastatic properties identifies TAOK3 as a key contributor to a cancer stem cell-like phenotype

Pancreatic cancer remains an incurable condition. Its progression is driven, in part, by subsets of cancer cells that evade the cytotoxic effects of conventional chemotherapies. These cells are often low-cycling, multidrug resistant, and adopt a stem cell–like phenotype consistent with the concept of cancer stem cells (CSC). To identify drugs impacting on tumor-promoting CSCs, we performed a differential high-throughput drug screen in pancreatic cancer cells cultured in traditional (2D) monolayers versus three-dimensional (3D) spheroids which replicate key elements of the CSC model. Among the agents capable of killing cells cultured in both formats was a 1H-benzo[d]imidazol-2-amine–based inhibitor of IL2-inducible T-cell kinase (ITK; NCGC00188382, inhibitor #1) that effectively mediated growth inhibition and induction of apoptosis in vitro, and suppressed cancer progression and metastasis formation in vivo. An examination of this agent's polypharmacology via in vitro and in situ phosphoproteomic profiling demonstrated an activity profile enriched for mediators involved in DNA damage repair. Included was a strong inhibitory potential versus the thousand-and-one amino acid kinase 3 (TAOK3), CDK7, and aurora B kinases. We found that cells grown under CSC-enriching spheroid conditions are selectively dependent on TAOK3 signaling. Loss of TAOK3 decreases colony formation, expression of stem cell markers, and sensitizes spheroids to the genotoxic effect of gemcitabine, whereas overexpression of TAOK3 increases stem cell traits including tumor initiation and metastasis formation. By inactivating multiple components of the cell-cycle machinery in concert with the downregulation of key CSC signatures, inhibitor #1 defines a distinctive strategy for targeting pancreatic cancer cell populations

DC mobilization from the skin requires docking to immobilized CCL21 on lymphatic endothelium and intralymphatic crawling

Dermal DC mobilization requires docking to CCL21 on lymphatic endotheliu

Endogenous TNF alpha orchestrates the trafficking of neutrophils into and within lymphatic vessels during acute inflammation

This work was supported by funds from Arthritis Research UK (19913 to M.-B.V.) and the Wellcome Trust (098291/Z/12/Z to S.N.). S. A. was supported by a QMUL Principal’s Award PhD Studentship