Nicotiana benthamiana Kunitz peptidase inhibitor-like protein involved in chloroplast-to-nucleus regulatory pathway in plant-virus interaction.

Plant viruses use a variety of strategies to infect their host. During infection, viruses cause symptoms of varying severity, which are often associated with altered leaf pigmentation due to structural and functional damage to chloroplasts that are affected by viral proteins. Here we demonstrate that Nicotiana benthamiana Kunitz peptidase inhibitor-like protein (KPILP) gene is induced in response to potato virus X (PVX) infection. Using reverse genetic approach, we have demonstrated that KPILP downregulates expression of LHCB1 and LHCB2 genes of antenna light-harvesting complex proteins, HEMA1 gene encoding glutamyl-tRNA reductase, which participates in tetrapyrrole biosynthesis, and RBCS1A gene encoding RuBisCO small subunit isoform involved in the antiviral immune response. Thus, KPILP is a regulator of chloroplast retrograde signaling system during developing PVX infection. Moreover, KPILP was demonstrated to affect carbon partitioning: reduced glucose levels during PVX infection were associated with KPILP upregulation. Another KPILP function is associated with plasmodesmata permeability control. Its ability to stimulate intercellular transport of reporter 2xGFP molecules indicates that KPILP is a positive plasmodesmata regulator. Moreover, natural KPILP glycosylation is indispensable for manifestation of this function. During PVX infection KPILP increased expression leads to the reduction of plasmodesmata callose deposition. These results could indicate that KPILP affects plasmodesmata permeability via callose-dependent mechanism. Thus, virus entering a cell and starting reproduction triggers KPILP expression, which leads to downregulation of nuclear-encoded chloroplast genes associated with retrograde signaling, reduction in photoassimilates accumulation and increase in intercellular transport, creating favorable conditions for reproduction and spread of viral infection.

Inhibition of Neutrophil Secretion Upon Adhesion as a Basis for the Anti-Inflammatory Effect of the Tricyclic Antidepressant Imipramine.

Recent studies demonstrate the involvement of inflammatory processes in the development of depression and the anti-inflammatory effects of antidepressants. Infiltration and adhesion of neutrophils to nerve tissues and their aggressive secretion are considered as possible causes of inflammatory processes in depression. We studied the effect of the antidepressant imipramine on the adhesion and accompanied secretion of neutrophils under control conditions and in the presence of lipopolysaccharides (LPS). As a model of integrin-dependent neutrophil infiltration into tissues, we used integrin-dependent adhesion of neutrophils to the fibronectin-coated substrate. Imipramine inhibited neutrophil adhesion and concomitant secretion of proteins, including matrix metalloproteinase 9 (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL), which modify the extracellular matrix and basement membranes required for cell migration. Imipramine also significantly and selectively blocked the release of the free amino acid hydroxylysine, a product of lysyl hydroxylase, an enzyme that affects the organization of the extracellular matrix by modifying collagen lysine residues. In contrast, imipramine enhanced the release of ROS by neutrophils during adhesion to fibronectin and stimulated apoptosis. The anti-inflammatory effect of imipramine may be associated with the suppression of neutrophil infiltration and their adhesion to nerve tissues by inhibiting the secretion of neutrophils, which provides these processes.

Synthetic Hexanucleotides as a Tool to Overcome Excessive Neutrophil Activation Caused by CpG-Containing Oligonucleotides.

Mimicking bacterial DNA, synthetic CpG-containing oligodeoxyribonucleotides (CpG-ODNs) have a powerful immunomodulatory potential. Their practical application is mainly associated with the production of vaccines, where they are used as adjuvants, as well as in local antimicrobial therapy. CpG-ODNs act on a wide variety of immune cells, including neutrophilic granulocytes. On the one hand, the stimulatory effect provides both the direct implementation of their antimicrobial and fungicidal mechanisms, and an avalanche-like strengthening of the immune signal due to interaction with other participants in the immune process. On the other hand, hyperactivation of neutrophilic granulocytes can have negative consequences. In particular, the formation of unreasonably high amounts of reactive oxygen species leads to tissue damages and, as a consequence, a spontaneous aggravation and prolongation of the inflammatory process. Under physiological conditions, a large number of DNA fragments are present in inflammation foci: both of microbial and self-tissue origin. We investigated effects of several short modified hexanucleotides on the main indicators of neutrophil activation, as well as their influence on the immunomodulatory activity of known synthetic CpG-ODNs. The results obtained show that short oligonucleotides partially inhibit the prooxidant effect of synthetic CpG-ODNs without significantly affecting the ability of the latter to overcome bacteria-induced pro-survival effects on neutrophilic granulocytes.

Synthetic CpG oligonucleotides as potential modulators of neutrophil survival in PAMP-associated inhibition of apoptosis.

We studied the effects of a synthetic CpG oligonucleotide (CpG ODN2006) on polymorphonuclear leukocyte (PMNL, neutrophil) survival and oxidant status. CpG ODN2006 showed a dose-dependent effect on the apoptosis of resting neutrophils. Without affecting the viability of resting cells, low concentrations of CpG ODN2006 interfered with Salmonella typhimurium-mediated viability prolongation and increased neutrophil apoptosis to control levels. CpG ODN2006 stimulated neutrophil apoptosis by enhancing ROS generation. Even small doses of ODN could induce the production of intracellular superoxide anions. The high superoxide reactogenicity, including with respect to nitrogen oxide, led to increased levels of intracellular ROS and RNS, which ultimately caused apoptosis. The pro-oxidant effect of low concentrations of CpG ODN2006 was not sufficient to trigger irreversible pro-apoptotic mechanisms. However, the sensitivity of PMNLs to ODN2006, a modulator of apoptosis, increased significantly under conditions of infectious inflammation. Inactivated S. typhimurium proved to be suitable for simulating inflammatory conditions in vitro.

Erratum to "Mold Alkaloid Cytochalasin D Modifies the Morphology and Secretion of fMLP-, LPS-, or PMA-Stimulated Neutrophils upon Adhesion to Fibronectin".

[This corrects the article DOI: 10.1155/2017/4308684.].

Mold Alkaloid Cytochalasin D Modifies the Morphology and Secretion of fMLP-, LPS-, or PMA-Stimulated Neutrophils upon Adhesion to Fibronectin.

Neutrophils play an essential role in innate immunity due to their ability to migrate into infected tissues and kill microbes with bactericides located in their secretory granules. Neutrophil transmigration and degranulation are tightly regulated by actin cytoskeleton. Invading pathogens produce alkaloids that cause the depolymerization of actin, such as the mold alkaloid cytochalasin D. We studied the effect of cytochalasin D on the morphology and secretion of fMLP-, LPS-, or PMA-stimulated human neutrophils upon adhesion to fibronectin. Electron microscopy showed that the morphology of the neutrophils adherent to fibronectin in the presence of various stimuli differed. But in the presence of cytochalasin D, all stimulated neutrophils exhibited a uniform nonspread shape and developed thread-like membrane tubulovesicular extensions (cytonemes) measuring 200 nm in diameter. Simultaneous detection of neutrophil secretory products by mass spectrometry showed that all tested stimuli caused the secretion of MMP-9, a key enzyme in the neutrophil migration. Cytochalasin D impaired the MMP-9 secretion but initiated the release of cathepsin G and other granular bactericides, proinflammatory agents. The release of bactericides apparently occurs through the formation, shedding, and lysis of cytonemes. The production of alkaloids which modify neutrophil responses to stimulation via actin depolymerization may be part of the strategy of pathogen invasion.

Inhibition of the GTPase dynamin or actin depolymerisation initiates outward plasma membrane tubulation/vesiculation (cytoneme formation) in neutrophils.

BACKGROUND INFORMATION: In a previous study, we demonstrated that human neutrophils can develop membrane tubulovesicular extensions (TVEs) that are 160-250 nm in width and several micrometres long. These extensions, or cytonemes, are capable of establishing long-range contacts with other cells or bacteria. Cytonemes consist of membrane tubules and vesicles of a uniform diameter aligned in a row. The mechanism of membrane tubulation/vesiculation to form cytonemes remains unknown. Upon endocytosis, the GTPase dynamin and an intact actin cytoskeleton are required for endocytic vesicles scission from the plasma membrane. RESULTS: We examined the effects of dynasore (a dynamin specific inhibitor), and of cytochalasin D and latrunculin A (actin cytoskeleton disruption agents), on cytoneme formation in neutrophils. Scanning and transmission electron microscopy were used to observe cytoneme formation. High-performance chromatography and mass spectrometry were used to estimate the protein composition of the cytonemes. In neutrophils, dynasore and cytochalasin D or latrunculin A initiated the formation of tubular cytonemes that were similar in diameter and composition. The formation of cytonemes in cells treated with cytochalasin D was accompanied by the appearance of tubular invaginations of the same diameter on the plasma membrane of neutrophils. The formation of dynasore- or cytochalasin D-induced cytonemes, however, was blocked by the nitric oxide (NO) synthases inhibitor l-NAME, indicating that NO is involved in cytoneme development. Proteome analysis indicated that dynasore- or cytochalasin D-induced cytonemes are secretory protrusions that contain neutrophil bactericides along with cytoplasmic proteins, such as glycolytic enzymes and actin cytoskeleton components. CONCLUSIONS: Inhibition of dynamin with dynasore or actin depolymerisation with cytochalasin D or latrunculin A might impair the membrane fusion/fission events that are required for the separation of secretory vesicles from the plasma membrane and from each other. As a result, the secretory process extends from the cells as membrane TVEs or cytonemes. Modification of secretion gives neutrophils the possibility to communicate with other cells over distance via highly adhesive cellular secretory protrusions (cytonemes). Cytonemes deliver their membrane-packed content exactly to the destination without dilution and without harm to the surrounding tissues.