Exosomes mediate LTB4 release during neutrophil chemotaxis.

Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies, which, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in an LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments.

Repeated Dose Toxicity Study and Developmental and Reproductive Toxicology Studies of a Respiratory Syncytial Virus Candidate Vaccine in Rabbits and Rats.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections, and vaccines are needed to treat young children and older adults. One of GSK's candidate vaccines for RSV contains recombinant RSVPreF3 protein maintained in the prefusion conformation. The differences in immune function of young children and older adults potentially require different vaccine approaches. For young children, anti-RSV immunity can be afforded during the first months of life by vaccinating the pregnant mother during the third trimester with unadjuvanted RSVPreF3, which results in protection of the infant due to the transplacental passage of anti-RSV maternal antibodies. For older adults with a waning immune response, the approach is to adjuvant the RSVPreF3 vaccine with AS01 to elicit a more robust immune response.The local and systemic effects of biweekly intramuscular injections of the RSVPreF3 vaccine (unadjuvanted, adjuvanted with AS01, or coadministered with a diphtheria-tetanus-acellular pertussis vaccine) was tested in a repeated dose toxicity study in rabbits. After three intramuscular doses, the only changes observed were those commonly related to a vaccine-elicited inflammatory reaction. Subsequently, the effects of unadjuvanted RSVPreF3 vaccine on female fertility, embryo-fetal, and postnatal development of offspring were evaluated in rats and rabbits. There were no effects on pregnancy, delivery, lactation, or the pre- and postnatal development of offspring.In conclusion, the RSVPreF3 vaccine was well-tolerated locally and systemically and was not associated with any adverse effects on female reproductive function or on the pre- and postnatal growth and development of offspring.

A role for keratins in supporting mitochondrial organization and function in skin keratinocytes.

Mitochondria fulfill essential roles in ATP production, metabolic regulation, calcium signaling, generation of reactive oxygen species (ROS), and additional determinants of cellular health. Recent studies have highlighted a role for mitochondria during cell differentiation, including in skin epidermis. The observation of oxidative stress in keratinocytes from Krt16 null mouse skin, a model for pachyonychia congenita (PC)-associated palmoplantar keratoderma, prompted us to examine the role of Keratin (K) 16 protein and its partner K6 in regulating the structure and function of mitochondria. Electron microscopy revealed major anomalies in mitochondrial ultrastructure in late stage, E18.5, Krt6a/Krt6b null embryonic mouse skin. Follow-up studies utilizing biochemical, metabolic, and live imaging readouts showed that, relative to controls, skin keratinocytes null for Krt6a/Krt6b or Krt16 exhibit elevated ROS, reduced mitochondrial respiration, intracellular distribution differences, and altered movement of mitochondria within the cell. These findings highlight a novel role for K6 and K16 in regulating mitochondrial morphology, dynamics, and function and shed new light on the causes of oxidative stress observed in PC and related keratin-based skin disorders.

Non-canonical processes that shape the cell migration landscape.

Migration is a vital, intricate, and multi-faceted process that involves the entire cell, entails the integration of multiple external cues and, at times, necessitates high-level coordination among fields of cells that can be physically attached or not, depending on the physiological setting. Recent advances have highlighted the essential role of cellular components that have not been traditionally considered when studying cell migration. This review details how much we recently learned by studying the role of intermediate filaments, the nucleus, extracellular vesicles, and mitochondria during cell migration.

Genetic manipulation of PLB-985 cells and quantification of chemotaxis using the underagarose assay.

Neutrophils are the most common leukocyte in human blood and are the first cells to respond to injury and infection. Improper neutrophil chemotaxis can have deleterious effects on human health, including autoimmune diseases, poor innate immune response, and cancer. Therefore, gaining a better understanding of the signaling pathways governing chemotactic responses in these cells is important. One of the main challenges of working with primary human neutrophils is their short lifespan (about 1 day), making genetic manipulations not feasible. PLB-985 cells, which are pluripotent hematopoietic cells that can easily be differentiated to neutrophil-like cells, are amenable to genetic manipulations, including the expression of fluorescently tagged proteins-of-interest (POI) and gene editing using the CRISPR/CAS9 system to delete genes-of-interest (GOI). The use of PLB-985 cells can therefore greatly facilitate our understanding of the molecular mechanisms governing neutrophil biology during chemotaxis and serve as a good system to complement results gained from pharmacological inhibition of primary neutrophils. To better study the role and localization of proteins during chemotaxis, the underagarose assay has become a widely used and quantitative assay for measuring several aspects of chemotaxis. The objective of this chapter is to provide protocols for (1) the generation of genetically altered PLB-985 cell lines, (2) the set-up of an underagarose chemotaxis assay, and (3) the analysis of cell movement in chemotactic gradients from an underagarose experiment.

Extracellular vesicles direct migration by synthesizing and releasing chemotactic signals.

Chemotactic signals are relayed to neighboring cells through the secretion of additional chemoattractants. We previously showed in Dictyostelium discoideum that the adenylyl cyclase A, which synthesizes the chemoattractant cyclic adenosine monophosphate (cAMP), is present in the intraluminal vesicles of multivesicular bodies (MVBs) that coalesce at the back of cells. Using ultrastructural reconstructions, we now show that ACA-containing MVBs release their contents to attract neighboring cells. We show that the released vesicles are capable of directing migration and streaming and are central to chemotactic signal relay. We demonstrate that the released vesicles not only contain cAMP but also can actively synthesize and release cAMP to promote chemotaxis. Through proteomic, pharmacological, and genetic approaches, we determined that the vesicular cAMP is released via the ABCC8 transporter. Together, our findings show that extracellular vesicles released by Ddiscoideum cells are functional entities that mediate signal relay during chemotaxis and streaming.

The role of the LTB4-BLT1 axis in chemotactic gradient sensing and directed leukocyte migration.

Directed leukocyte migration is a hallmark of inflammatory immune responses. Leukotrienes are derived from arachidonic acid and represent a class of potent lipid mediators of leukocyte migration. In this review, we summarize the essential steps leading to the production of LTB4 in leukocytes. We discuss the recent findings on the exosomal packaging and transport of LTB4 in the context of chemotactic gradients formation and regulation of leukocyte recruitment. We also discuss the dynamic roles of the LTB4 receptors, BLT1 and BLT2, in mediating chemotactic signaling in leukocytes and contrast them to other structurally related leukotrienes that bind to distinct GPCRs. Finally, we highlight the specific roles of the LTB4-BLT1 axis in mediating signal-relay between chemotaxing neutrophils and its potential contribution to a wide variety of inflammatory conditions including tumor progression and metastasis, where LTB4 is emerging as a key signaling component.

Modeling neutrophil migration in dynamic chemoattractant gradients: assessing the role of exosomes during signal relay.

Migrating cells often exhibit signal relay, a process in which cells migrating in response to a chemotactic gradient release a secondary chemoattractant to enhance directional migration. In neutrophils, signal relay toward the primary chemoattractant N--formylmethionyl-leucyl-phenylalanine (fMLP) is mediated by leukotriene B4 (LTB4). Recent evidence suggests that the release of LTB4 from cells occurs through packaging in exosomes. Here we present a mathematical model of neutrophil signal relay that focuses on LTB4 and its exosome-mediated secretion. We describe neutrophil chemotaxis in response to a combination of a defined gradient of fMLP and an evolving gradient of LTB4, generated by cells in response to fMLP. Our model enables us to determine the gradient of LTB4 arising either through directed secretion from cells or through time-varying release from exosomes. We predict that the secondary release of LTB4 increases recruitment range and show that the exosomes provide a time delay mechanism that regulates the development of LTB4 gradients. Additionally, we show that under decaying primary gradients, secondary gradients are more stable when secreted through exosomes as compared with direct secretion. Our chemotactic model, calibrated from observed responses of cells to gradients, thereby provides insight into chemotactic signal relay in neutrophils during inflammation.

Exosomes Mediate LTB4 Release during Neutrophil Chemotaxis.

Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies that, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in a LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments.

New paradigms in the establishment and maintenance of gradients during directed cell migration.

Directional guidance of migrating cells is relatively well explored in the reductionist setting of cell culture experiments. Here spatial gradients of chemical cues as well as gradients of mechanical substrate characteristics prove sufficient to attract single cells as well as their collectives. How such gradients present and act in the context of an organism is far less clear. Here we review recent advances in understanding how guidance cues emerge and operate in complex physiological settings.

Cell migration: sinking in a gradient.

How chemoattractant gradients form and persist in complex tissues is a key question in cell migration. Two studies now show that CXCR7 acts as a sink in the migrating zebrafish lateral line primordium to generate SDF1 gradients.

Biotin-conjugated tumour-targeting photocytotoxic iron(III) complexes.

Iron(III) complexes [FeL(B)] (1-4) of a tetradentate phenolate-based ligand (H3L) and biotin-conjugated dipyridophenazine bases (B), viz. 7-aminodipyrido [3,2-a:2',3'-c]-phenazine (dppza in 1), (N-dipyrido[3,2-a:2',3'-c]-phenazino)amidobiotin (dppzNB in 2), dipyrido [3,2-a:2',3'-c]-phenazine-11-carboxylic acid (dppzc in 3) and 2-((2-biotinamido)ethyl) amido-dipyrido[3,2-a:2',3'-c]-phenazine (dppzCB in 4) are prepared, characterized and their interaction with streptavidin and DNA and their photocytotoxicity and cellular uptake in various cells studied. The high-spin iron(III) complexes display Fe(III)/Fe(II) redox couple near -0.7 V versus saturated calomel electrode in dimethyl sulfoxide-0.1 M tetrabutylammonium perchlorate. The complexes show non-specific interaction with DNA as determined from the binding studies. Complexes with appended biotin moiety show similar binding to streptavidin as that of free biotin, suggesting biotin conjugation to dppz does not cause any loss in its binding affinity to streptavidin. The photocytotoxicity of the complexes is tested in HepG2, HeLa and HEK293 cell lines. Complex 2 shows higher photocytotoxicity in HepG2 cells than in HeLa or HEK293, forming reactive oxygen species. This effect is attributed to the presence of overexpressed sodium-dependent multi-vitamin transporters in HepG2 cells. Microscopic studies in HepG2 cells show internalization of the biotin complexes 2 and 4 essentially occurring by receptor-mediated endocytosis, which is similar to that of native biotin and biotin fluorescein isothiocyanate conjugate.

The antibodies against the computationally designed mimic of the glycoprotein hormone receptor transmembrane domain provide insights into receptor activation and suppress the constitutively activated receptor mutants.

The exoloops of glycoprotein hormone receptors (GpHRs) transduce the signal generated by the ligand-ectodomain interactions to the transmembrane helices either through direct hormonal contact and/or by modulating the interdomain interactions between the hinge region (HinR) and the transmembrane domain (TMD). The ligand-induced conformational alterations in the HinRs and the interhelical loops of luteinizing hormone receptor/follicle stimulating hormone receptor/thyroid stimulating hormone receptor were mapped using exoloop-specific antibodies generated against a mini-TMD protein designed to mimic the native exoloop conformations that were created by joining the thyroid stimulating hormone receptor exoloops constrained through helical tethers and library-derived linkers. The antibody against the mini-TMD specifically recognized all three GpHRs and inhibited the basal and hormone-stimulated cAMP production without affecting hormone binding. Interestingly, binding of the antibody to all three receptors was abolished by prior incubation of the receptors with the respective hormones, suggesting that the exoloops are buried in the hormone-receptor complexes. The antibody also suppressed the high basal activities of gain-of-function mutations in the HinRs, exoloops, and TMDs such as those involved in precocious puberty and thyroid toxic adenomas. Using the antibody and point/deletion/chimeric receptor mutants, we demonstrate that changes in the HinR-exoloop interactions play an important role in receptor activation. Computational analysis suggests that the mini-TMD antibodies act by conformationally locking the transmembrane helices by means of restraining the exoloops and the juxta-membrane regions. Using GpHRs as a model, we describe a novel computational approach of generating soluble TMD mimics that can be used to explain the role of exoloops during receptor activation and their interplay with TMDs.

The hinge region of human thyroid-stimulating hormone (TSH) receptor operates as a tunable switch between hormone binding and receptor activation.

The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding to activation of downstream effecters is not clearly understood. In the present study, agonistic (311.62) and antagonistic (311.87) monoclonal antibodies (MAbs) directed against the TSH receptor extracellular domain were used to elucidate role of the hinge region in receptor activation. MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of Thyroid stimulating hormone (TSH) binding. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), this study confirms the tethered inverse agonistic role played by the hinge region and maps the interactions between TSHR hinge region and exoloops responsible for maintenance of the receptor in its basal state. Mechanistic studies on the antibody-receptor interactions suggest that MAb 311.87 is an allosteric insurmountable antagonist and inhibits initiation of the hormone induced conformational changes in the hinge region, whereas MAb 311.62 acts as a partial agonist that recognizes a conformational epitope critical for coupling of hormone binding to receptor activation. The hinge region, probably in close proximity with the α-subunit in the hormone-receptor complex, acts as a tunable switch between hormone binding and receptor activation.

Insights into differential modulation of receptor function by hinge region using novel agonistic lutropin receptor and inverse agonistic thyrotropin receptor antibodies.

We report two antibodies, scFv 13B1 and MAb PD1.37, against the hinge regions of LHR and TSHR, respectively, which have similar epitopes but different effects on receptor function. While neither of them affected hormone binding, with marginal effects on hormone response, scFv 13B1 stimulated LHR in a dose-dependent manner, whereas MAb PD1.37 acted as an inverse agonist of TSHR. Moreover, PD1.37 could decrease the basal activity of hinge region CAMs, but had varied effects on those present in ECLs, whereas 13B1 was refractory to any CAMs in LHR. Using truncation mutants and peptide phage display, we compared the differential roles of the hinge region cysteine box-2/3 as well as the exoloops in the activation of these two homologus receptors.

Docking and free energy simulations to predict conformational domains involved in hCG-LH receptor interactions using recombinant antibodies.

Single chain fragment variables (ScFvs) have been extensively employed in studying the protein-protein interactions. ScFvs derived from phage display libraries have an additional advantage of being generated against a native antigen, circumventing loss of information on conformational epitopes. In the present study, an attempt has been made to elucidate human chorionic gonadotropin (hCG)-luteinizing hormone (LH) receptor interactions by using a neutral and two inhibitory ScFvs against hCG. The objective was to dock a computationally derived model of these ScFvs onto the crystal structure of hCG and understand the differential roles of the mapped epitopes in hCG-LH receptor interactions. An anti-hCG ScFv, whose epitope was mapped previously using biochemical tools, served as the positive control for assessing the quality of docking analysis. To evaluate the role of specific side chains at the hCG-ScFv interface, binding free energy as well as residue interaction energies of complexes in solution were calculated using molecular mechanics Poisson-Boltzmann/surface area method after performing the molecular dynamic simulations on the selected hCG-ScFv models and validated using biochemical and SPR analysis. The robustness of these calculations was demonstrated by comparing the theoretically determined binding energies with the experimentally obtained kinetic parameters for hCG-ScFv complexes. Superimposition of hCG-ScFv model onto a model of hCG complexed with the 51-266 residues of LH receptor revealed importance of the residues previously thought to be unimportant for hormone binding and response. This analysis provides an alternate tool for understanding the structure-function analysis of ligand-receptor interactions.

Remarkable photocytotoxicity in hypoxic HeLa cells by a dipyridophenazine copper(II) Schiff base thiolate.

Copper(II) complexes [Cu(satp)(L)] (1-3) of a Schiff base thiolate (salicylidene-2-aminothiophenol, H2satp) and phenanthroline bases (L), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq in 2) and dipyrido[3,2-a:2',3'-c]phenazine (dppz in 3), were prepared, characterized and their anaerobic DNA photocleavage activity and hypoxic photocytotoxicity studied. The redox active complexes show the Cu(II)-Cu(I) couple near -0.5 V for 1 and near 0.0 V vs. SCE (saturated calomel electrode) for 2 and 3. The one-electron paramagnetic complexes (~1.85 µB) are avid DNA binders giving Kb values within 1.0×10(5)-8.0×10(5) M(-1). Thermal melting and viscosity data along with molecular docking calculations suggest DNA groove and/or partial intercalative binding of the complexes. The complexes show anaerobic DNA cleavage activity in red light under argon via type-I pathway, while DNA photocleavage in air proceeds via hydroxyl radical pathway. The DFT (density functional theory) calculations reveal a thyil radical pathway for the anaerobic DNA photocleavage activity and suggest the possibility of generation of a transient copper(I) species due to bond breakage between the copper and sulfur to generate the thyil radical. An oxidation of the copper(I) species is likely by oxygen in an aerobic medium or by the buffer medium in an anaerobic condition. Complex 3 exhibits significant photocytotoxicity in HeLa cells (IC50=8.3(±1.0) µM) in visible light, while showing lower dark toxicity (IC50=17.2(±1.0) µM). A significant reduction in the dark toxicity is observed under hypoxic cellular conditions (IC50=30.0(±1.0) µM in dark), while retaining its photocytotoxicity (IC50=8.0(±1.0) µM).

Structure-activity relationship of photocytotoxic iron(III) complexes of modified dipyridophenazine ligands.

Iron(III) complexes [FeL(B)] (1-5) of a tetradentate trianionic phenolate-based ligand (L) and modified dipyridophenazine bases (B), namely, dipyrido-6,7,8,9-tetrahydrophenazine (dpqC in 1), dipyrido[3,2-a:2',3'-c]phenazine-2-carboxylic acid (dppzc in 2), dipyrido[3,2-a:2',3'-c]phenazine-11-sulfonic acid (dppzs in 3), 7-aminodipyrido[3,2-a:2',3'-c]phenazine (dppza in 4) and benzo[i]dipyrido[3,2-a:2',3'-c]phenazine (dppn in 5), have been synthesized and their photocytotoxic properties studied along with their dipyridophenazine analogue (6). The complexes have a five electron paramagnetic iron(III) center, and the Fe(III)/Fe(II) redox couple appears at about -0.69 V versus SCE in DMF-0.1 M TBAP. The physicochemical data also suggest that the complexes possess similar structural features as that of its parent complex [FeL(dppz)] with FeO3N3 coordination in a distorted octahedral geometry. The DNA-complex and protein-complex interaction studies have revealed that the complexes interact favorably with the biomolecules, the degree of which depends on the nature of the substituents present on the dipyridophenazine ring. Photocleavage of pUC19 DNA by the complexes has been studied using visible light of 476, 530, and 647 nm wavelengths. Mechanistic investigations with inhibitors show formation of HO(•) radicals via a photoredox pathway. Photocytotoxicity study of the complexes in HeLa cells has shown that the dppn complex (5) is highly active in causing cell death in visible light with sub micromolar IC(50) value. The effect of substitutions and the planarity of the phenazine moiety on the cellular uptake are quantified by determining the total cellular iron content using the inductively coupled plasma-optical emission spectrometry (ICP-OES) technique. The cellular uptake increases marginally with an increase in the hydrophobicity of the dipyridophenazine ligands whereas complex 3 with dppzs shows very high uptake. Insights into the cell death mechanism by the dppn complex 5, obtained through DAPI nuclear staining in HeLa cells, reveal a rapid programmed cell death mechanism following photoactivation of complex 5 with visible light. The effect of substituent on the DNA photocleavage activity of the complexes has been rationalized from the theoretical studies.

Cobalt(II) complexes of terpyridine bases as photochemotherapeutic agents showing cellular uptake and photocytotoxicity in visible light.

Cobalt(II) complexes of terpyridine bases [Co(L)2](ClO4)2 (1-3), where L is 4'-phenyl-2,2':6',2''-terpyridine (ph-tpy in 1), 4'-(9-anthracenyl)-2,2':6',2''-terpyridine (an-tpy in 2) and 4'-(1- pyrenyl)-2,2':6',2''-terpyridine (py-tpy in 3), are prepared and their photo-induced DNA and protein cleavage activity and photocytotoxic property in HeLa cells studied. The 1 : 2 electrolytic and three-electron paramagnetic complexes show a visible band near 550 nm in DMF-Tris-HCl buffer. The complexes 1-3 show emission spectral bands at 355, 421 and 454 nm, respectively, when excited at 287, 368 and 335 nm. The quantum yield values for 1-3 in DMF-H2O (2 : 1 v/v) are 0.025, 0.060 and 0.28, respectively. The complexes are redox active in DMF-0.1 M TBAP. The Co(III)-Co(II) and Co(II)-Co(I) couples appear as quasi-reversible cyclic voltammetric responses near 0.2 and -0.7 V vs. SCE, respectively. Complexes 2 and 3 are avid binders to calf thymus DNA giving K(b) value of ∼106 M⁻¹. The complexes show chemical nuclease activity. Complexes 2 and 3 exhibit oxidative cleavage of pUC19 DNA in UV-A and visible light. The DNA photocleavage reaction of 3 at 365 nm shows formation of singlet oxygen and hydroxyl radical species, while only hydroxyl radical formation is evidenced in visible light. Complexes 2 and 3 show non-specific photo-induced bovine serum albumin protein cleavage activity at 365 nm. The an-tpy and py-tpy complexes exhibit significant photocytotoxicity in HeLa cervical cancer cells on exposure to visible light giving IC50 values of 24.2 and 7.6 µM, respectively. Live cell imaging study shows accumulation of the complexes in the cytosol of HeLa cancer cells.