Correction: The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway.

[This corrects the article DOI: 10.1371/journal.pbio.1001090.].

Anomalous diffusion and asymmetric tempering memory in neutrophil chemotaxis.

The motility of neutrophils and their ability to sense and to react to chemoattractants in their environment are of central importance for the innate immunity. Neutrophils are guided towards sites of inflammation following the activation of G-protein coupled chemoattractant receptors such as CXCR2 whose signaling strongly depends on the activity of Ca2+ permeable TRPC6 channels. It is the aim of this study to analyze data sets obtained in vitro (murine neutrophils) and in vivo (zebrafish neutrophils) with a stochastic mathematical model to gain deeper insight into the underlying mechanisms. The model is based on the analysis of trajectories of individual neutrophils. Bayesian data analysis, including the covariances of positions for fractional Brownian motion as well as for exponentially and power-law tempered model variants, allows the estimation of parameters and model selection. Our model-based analysis reveals that wildtype neutrophils show pure superdiffusive fractional Brownian motion. This so-called anomalous dynamics is characterized by temporal long-range correlations for the movement into the direction of the chemotactic CXCL1 gradient. Pure superdiffusion is absent vertically to this gradient. This points to an asymmetric 'memory' of the migratory machinery, which is found both in vitro and in vivo. CXCR2 blockade and TRPC6-knockout cause tempering of temporal correlations in the chemotactic gradient. This can be interpreted as a progressive loss of memory, which leads to a marked reduction of chemotaxis and search efficiency of neutrophils. In summary, our findings indicate that spatially differential regulation of anomalous dynamics appears to play a central role in guiding efficient chemotactic behavior.

Cell type specific gene expression profiling reveals a role for complement component C3 in neutrophil responses to tissue damage.

Tissue damage induces rapid recruitment of leukocytes and changes in the transcriptional landscape that influence wound healing. However, the cell-type specific transcriptional changes that influence leukocyte function and tissue repair have not been well characterized. Here, we employed translating ribosome affinity purification (TRAP) and RNA sequencing, TRAP-seq, in larval zebrafish to identify genes differentially expressed in neutrophils, macrophages, and epithelial cells in response to wounding. We identified the complement pathway and c3a.1, homologous to the C3 component of human complement, as significantly increased in neutrophils in response to wounds. c3a.1-/- zebrafish larvae have impaired neutrophil directed migration to tail wounds with an initial lag in recruitment early after wounding. Moreover, c3a.1-/- zebrafish larvae have impaired recruitment to localized bacterial infections and reduced survival that is, at least in part, neutrophil mediated. Together, our findings support the power of TRAP-seq to identify cell type specific changes in gene expression that influence neutrophil behavior in response to tissue damage.

Correction: The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway.

[This corrects the article DOI: 10.1371/journal.pbio.1001090.].

Chemokine Signaling and the Regulation of Bidirectional Leukocyte Migration in Interstitial Tissues.

Motile cells navigate through complex tissue environments that include both attractive and repulsive cues. In response to tissue wounding, neutrophils, primary cells of the innate immune response, exhibit bidirectional migration that is orchestrated by chemokines and their receptors. Although progress has been made in identifying signals that mediate the recruitment phase, the mechanisms that regulate neutrophil reverse migration remain largely unknown. Here, we visualize bidirectional neutrophil migration to sterile wounds in zebrafish larvae and identify specific roles for the chemokine receptors Cxcr1 and Cxcr2 in neutrophil recruitment to sterile injury and infection. Notably, we also identify Cxcl8a/Cxcr2 as a specific ligand-receptor pair that orchestrates neutrophil chemokinesis in interstitial tissues during neutrophil reverse migration and resolution of inflammation. Taken together, our findings identify distinct receptors that mediate bidirectional leukocyte motility during interstitial migration depending on the context and type of tissue damage in vivo.

The cleaved FAS ligand activates the Na(+)/H(+) exchanger NHE1 through Akt/ROCK1 to stimulate cell motility.

Transmembrane CD95L (Fas ligand) can be cleaved to release a promigratory soluble ligand, cl-CD95L, which can contribute to chronic inflammation and cancer cell dissemination. The motility signaling pathway elicited by cl-CD95L remains poorly defined. Here, we show that in the presence of cl-CD95L, CD95 activates the Akt and RhoA signaling pathways, which together orchestrate an allosteric activation of the Na(+)/H(+) exchanger NHE1. Pharmacologic inhibition of Akt or ROCK1 independently blocks the cl-CD95L-induced migration. Confirming these pharmacologic data, disruption of the Akt and ROCK1 phosphorylation sites on NHE1 decreases cell migration in cells exposed to cl-CD95L. Together, these findings demonstrate that NHE1 is a novel molecular actor in the CD95 signaling pathway that drives the cl-CD95L-induced cell migration through both the Akt and RhoA signaling pathways.

Redox and Src family kinase signaling control leukocyte wound attraction and neutrophil reverse migration.

Tissue damage induces early recruitment of neutrophils through redox-regulated Src family kinase (SFK) signaling in neutrophils. Redox-SFK signaling in epithelium is also necessary for wound resolution and tissue regeneration. How neutrophil-mediated inflammation resolves remains unclear. In this paper, we studied the interactions between macrophages and neutrophils in response to tissue damage in zebrafish and found that macrophages contact neutrophils and induce resolution via neutrophil reverse migration. We found that redox-SFK signaling through p22phox and Yes-related kinase is necessary for macrophage wound attraction and the subsequent reverse migration of neutrophils. Importantly, macrophage-specific reconstitution of p22phox revealed that macrophage redox signaling is necessary for neutrophil reverse migration. Thus, redox-SFK signaling in adjacent tissues is essential for coordinated leukocyte wound attraction and repulsion through pathways that involve contact-mediated guidance.

Neutrophil migration: moving from zebrafish models to human autoimmunity.

There has been a resurgence of interest in the neutrophil's role in autoimmune disease. Classically considered an early responder that dies at the site of inflammation, new findings using live imaging of embryonic zebrafish and other modalities suggest that neutrophils can reverse migrate away from sites of inflammation. These 'inflammation-sensitized' neutrophils, as well as the neutrophil extracellular traps and other products made by neutrophils in general, may have many implications for autoimmunity. Here, we review what is known about the role of neutrophils in three different autoimmune diseases: rheumatoid arthritis, systemic lupus erythematosus, and small vessel vasculitis. We then highlight recent findings related to several cytoskeletal regulators that guide neutrophil recruitment including Lyn, Rac2, and SHIP. Finally, we discuss how our improved understanding of the molecules that control neutrophil chemotaxis may impact our knowledge of autoimmunity.

CD95L cell surface cleavage triggers a prometastatic signaling pathway in triple-negative breast cancer.

Triple-negative breast cancers (TNBC) lacking estrogen and progesterone receptors and HER2 amplification have a relatively high risk of metastatic dissemination, but the mechanistic basis for this risk is not understood. Here, we report that serum levels of CD95 ligand (CD95L) are higher in patients with TNBC than in other patients with breast cancer. Metalloprotease-mediated cleavage of CD95L expressed by endothelial cells surrounding tumors generates a gradient that promotes cell motility due to the formation of an unconventional CD95-containing receptosome called the motility-inducing signaling complex. The formation of this complex was instrumental for Nox3-driven reactive oxygen species generation. Mechanistic investigations revealed a Yes-Orai1-EGFR-PI3K pathway that triggered migration of TNBC cells exposed to CD95L. Our findings establish a prometastatic function for metalloprotease-cleaved CD95L in TNBCs, revisiting its role in carcinogenesis.

The CD95 signaling pathway: To not die and fly.

Our recent findings indicate that cells exposed to transmembrane (m-CD95L) or metalloprotease-cleaved CD95L (cl-CD95L) undergo a localized Ca(2+)entry that not only inhibits the initial steps of the CD95-mediated apoptotic signal but also promotes cell motility. Based on recent findings published on the non-apoptotic signals induced by CD95, we discuss how m-CD95L and cl-CD95L diverging by their stoichiometry could both contribute to the immune response by first recruiting activated T lymphocytes in the inflamed area and later by eliminating infected and transformed cells.

CD95-mediated cell signaling in cancer: mutations and post-translational modulations.

Apoptosis has emerged as a fundamental process important in tissue homeostasis, immune response, and during development. CD95 (also known as Fas), a member of the tumor necrosis factor receptor (TNF-R) superfamily, has been initially cloned as a death receptor. Its cognate ligand, CD95L, is mainly found at the plasma membrane of activated T-lymphocytes and natural killer cells where it contributes to the elimination of transformed and infected cells. According to its implication in the immune homeostasis and immune surveillance, and since several malignant cells of various histological origins exhibit loss-of-function mutations, which cause resistance towards the CD95-mediated apoptotic signal, CD95 has been classified as a tumor suppressor gene. Nevertheless, this assumption has been recently challenged, as in certain pathophysiological contexts, CD95 engagement transmits non-apoptotic signals that promote inflammation, carcinogenesis or liver/peripheral nerve regeneration. The focus of this review is to discuss these apparent contradictions of the known function(s) of CD95.

The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway.

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca²âº/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells.

Actin-independent exclusion of CD95 by PI3K/AKT signalling: implications for apoptosis.

The immune system eliminates infected or transformed cells through the activation of the death receptor CD95. CD95 engagement drives the recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates and activates initiator caspases-8 and -10. The CD95-mediated apoptotic signal relies on the capacity to form the CD95/FADD/caspases complex termed the death-inducing signalling complex (DISC). Cells are classified according to the magnitude of DISC formation as either type I (efficient DISC formation) or type II (inefficient). CD95 localised to lipid rafts in type I cells, whereas the death receptor was excluded from these domains in type II cells. Here, we show that inhibition of both PI3K class IA and serine-threonine kinase Akt in type II cells promoted the redistribution of CD95 into lipid rafts, DISC formation and the initiation of the apoptotic signal. Strikingly, these molecular events took place independently of CD95L and the actin cytoskeleton. Overall, these findings highlight that the oncogenic PI3K/Akt signalling pathway participates in maintaining cells in a type II phenotype by excluding CD95 from lipid rafts.

Gene expression profiling provides insights into pathways of oxaliplatin-related sinusoidal obstruction syndrome in humans.

Sinusoidal obstruction syndrome (SOS; formerly veno-occlusive disease) is a well-established complication of hematopoietic stem cell transplantation, pyrrolizidine alkaloid intoxication, and widely used chemotherapeutic agents such as oxaliplatin. It is associated with substantial morbidity and mortality. Pathogenesis of SOS in humans is poorly understood. To explore its molecular mechanisms, we used Affymetrix U133 Plus 2.0 microarrays to investigate the gene expression profile of 11 human livers with oxaliplatin-related SOS and compared it to 12 matched controls. Hierarchical clustering analysis showed that profiles from SOS and controls formed distinct clusters. To identify functional networks and gene ontologies, data were analyzed by the Ingenuity Pathway Analysis Tool. A total of 913 genes were differentially expressed in SOS: 613 being upregulated and 300 downregulated. Reverse transcriptase-PCR results showed excellent concordance with microarray data. Pathway analysis showed major gene upregulation in six pathways in SOS compared with controls: acute phase response (notably interleukin 6), coagulation system (Serpine1, THBD, and VWF), hepatic fibrosis/hepatic stellate cell activation (COL3a1, COL3a2, PDGF-A, TIMP1, and MMP2), and oxidative stress. Angiogenic factors (VEGF-C) and hypoxic factors (HIF1A) were upregulated. The most significant increase was seen in CCL20 mRNA. In conclusion, oxaliplatin-related SOS can be readily distinguished according to morphologic characteristics but also by a molecular signature. Global gene analysis provides new insights into mechanisms underlying chemotherapy-related hepatotoxicity in humans and potential targets relating to its diagnosis, prevention, and treatment. Activation of VEGF and coagulation (vWF) pathways could partially explain at a molecular level the clinical observations that bevacizumab and aspirin have a preventive effect in SOS.

Membrane-associated signaling in human B-lymphoma lines.

In B-non-Hodgkin lymphomas, Lyn and Cbp/PAG constitute the core of an oncogenic signalosome that captures the Phosphatidylinositol-3-kinase, the Spleen tyrosine kinase and the Signal transducer and activator of transcription-3 to generate pro-survival and proliferative signals. Lymphoma lines corresponding to follicular, mantle-cell and Burkitt-derived lymphomas display type-specific signalosome organizations that differentially activate PI3K, Syk and STAT3. In the follicular lymphoma line, PI3K, Syk and STAT3 were optimally activated upon association with the Lyn-Cbp/PAG signalosome, while in the Burkitt lymphoma-derived line, the association with Cbp/PAG and activation of PI3K were interfered with by the latent membrane proteins encoded by the Epstein-Barr virus. In the Jeko-1 mantle-cell line, a weak association of Syk with the Lyn-Cbp/PAG signalosome resulted in poor activation of Syk, but in those cells, as in the follicular and Burkitt-derived lines, efficient apoptosis induction by the Syk inhibitor R406 indicated that Syk is nonetheless an important prosurvival element and therefore a valuable therapeutic target. In all configurations described herein is the Lyn-Cbp/PAG signalosome independent of external signals and provides efficient means of activation for its associated lipid and protein kinases. In follicular and Burkitt-derived lines, Syk appears to be activated following binding to Cbp/PAG and no longer requires B-cell receptor-associated activation motifs for activation. Assessment of the different modalities of Lyn-Cbp/PAG signalosome organization could help in selecting the appropriate combination of kinase inhibitors to eliminate a particular type of lymphoma cells.

Soluble TNF, but not membrane TNF, is critical in LPS-induced hepatitis.

BACKGROUND & AIMS: : Bacillus Calmette-Guérin (BCG) infection causes hepatic injury following granuloma formation and secretion of cytokines which renders mice highly sensitive to endotoxin-mediated hepatotoxicity. Tumor necrosis factor (TNF) is required for granuloma formation and is one of the most important cytokines in liver injury. TNF inhibitors are effective therapies for inflammatory diseases. However, clinical use of non-selective TNF inhibitors is associated with an increased risk of infections. This work investigates the differential roles of soluble TNF (solTNF) and membrane TNF (memTNF) in BCG infection, BCG/LPS- and D-GALN/LPS-induced liver injury. METHODS: We have used both genetic and pharmacologic approaches and analyzed liver injury, TLR4, cytokine and iNOS activation induced by BCG, BCG/LPS and D-GALN/LPS. RESULTS: BCG infection-induced liver injury is seen in wild-type mice but not in TNF(-/-), memTNF knock-in (KI), and sTNFR1-Fc transgenic mice. Severity of BCG-induced liver injury is correlated with BCG-granuloma number and hepatic expression of TLR4 and iNOS. In addition, protection from liver damage caused by BCG/LPS or D-GALN/LPS administration was observed in TNF(-/-), memTNF KI and sTNFR1-Fc transgenic mice. To extend the genetic findings, we then evaluated whether selective pharmacological inhibition of solTNF by dominant-negative (DN)-TNF neutralization and non-selective inhibition of solTNF and memTNF by anti-TNF antibodies and etanercept (TNFR2-IgG1) can protect the mice from liver injury. Both selective and non-selective inhibition of solTNF protected mice from BCG/LPS and D-GALN/LPS-induced liver damage. CONCLUSIONS: These data suggest that memTNF is not mediating liver injury and that selective inhibition of solTNF sparing memTNF may represent a new therapeutic strategy to treat immune-mediated inflammatory liver diseases.

The sphingolipid-rich rafts of ALK+ lymphomas downregulate the Lyn-Cbp/PAG signalosome.

Human anaplastic lymphoma kinase (ALK) + lymphomas express the constitutively active ALK as a fusion protein that drives several survival pathways. The catalytic domain of the anaplastic receptor tyrosine kinase is frequently fused with the nuclear localization protein nucleophosmin but may also fuse with other proteins that associate it with other subcellular structures. Similarly to other B human lymphomas, ALK+ lymphomas express the Cbp/PAG adaptor protein and the non-receptor Lyn kinase in the plasma membrane. In the majority of human lymphomas, the Cbp/PAG adaptor and the Lyn kinase constitute an oncogenic signalosome that serves as a membrane anchor for other signaling enzymes and transcription factors. We show that ALK+ lymphoma membranes harbor sphingolipid-rich microdomains (rafts) in which Lyn is poorly active. However, Lyn activity and consequently Cbp/PAG tyrosine phosphorylation can be restored by extracting sphingolipids from ALK+ lymphoma plasma membranes. In the membrane environment of ALK+ lymphoma rafts, where the glycosphingolipid to signaling protein ratio is higher than in B-NHL rafts, the Lyn activity is suboptimal and does not allow the formation of an efficient Lyn-Cbp/PAG signalosome.

Phytochemicals as modulators of neoplastic phenotypes.

It is generally accepted that nutritional behaviors constitute decisive components of human health. Phytochemicals (small, nonenergetic molecules of vegetal origin) are overall inhibitory on the expression of gene products promoting proliferation and resistance to apoptosis. On the contrary, phytochemicals stimulate the synthesis of adaptive proteins that favor resistance to cellular stress (detoxifying and antioxidant enzymes). They are effective modulators that act synergistically on membrane, cytoplasmic and nuclear enzymatic reactions to dampen cellular hyperproliferation and hyperactivity, reequilibrate metabolic activity and promote apoptosis of genetically unstable cells. Despite important gaps in our knowledge regarding how phytochemicals interfere with cellular function in vivo, effective chemopreventive measures have shown that phytochemicals can be utilized to prevent cancer, and possibly to treat cancer patients as well. We review how phytochemicals exert their beneficial effects at the cellular level.

Oncogenic association of the Cbp/PAG adaptor protein with the Lyn tyrosine kinase in human B-NHL rafts.

B-non-Hodgkin lymphomas (B-NHLs) use a raft-associated signalosome made of the constitutively active Lyn kinase, the tyrosine phosphorylated Cbp/PAG adaptor, and tyrosine phosphorylated STAT3 transcription factor. No such "signalosome" is found in rafts of ALK(+) T lymphoma and Hodgkin-derived cell lines, despite similar Cbp/PAG, Lyn, and STAT3 expression and similar amounts of raft sphingolipids. Stable association of the signalosome with B-NHL rafts requires (1) a Lyn kinase (auto)phosphorylated in its regulatory and active site tyrosines, (2) a Cbp/PAG adaptor phosphorylated at tyrosine 317 and bound to Lyn SH2 via phosphotyrosine 299 and neighboring residues, and (3) a tyrosine phosphorylated STAT3 linked via SH2 to the regulatory, C-terminal tyrosine of Lyn. No Csk appears to be part of this B-NHL signalosome. An oncogenic role for Lyn was shown after exposure of B-NHL lines to Lyn inhibitors that prevented Lyn and Cbp/PAG phosphorylation, dissociated the signalosome from rafts, and eventually induced death. Cell death followed decreases in Lyn or Cbp/PAG expression levels in one mantle cell lymphoma line, but not in a Hodgkin-derived one. The Lyn-Cbp/PAG signalosome appears to control proliferation and survival in most B-NHLs and constitutes a therapeutic target in B-NHL cells that exhibit oncogenic "addiction" to the Lyn kinase.