Lyn is a redox sensor that mediates leukocyte wound attraction in vivo.

Tissue wounding induces the rapid recruitment of leukocytes. Wounds and tumours--a type of 'unhealed wound'--generate hydrogen peroxide (H(2)O(2)) through an NADPH oxidase (NOX). This extracellular H(2)O(2) mediates recruitment of leukocytes, particularly the first responders of innate immunity, neutrophils, to injured tissue. However, the sensor that neutrophils use to detect the redox state at wounds is unknown. Here we identify the Src family kinase (SFK) Lyn as a redox sensor that mediates initial neutrophil recruitment to wounds in zebrafish larvae. Lyn activation in neutrophils is dependent on wound-derived H(2)O(2) after tissue injury, and inhibition of Lyn attenuates neutrophil wound recruitment. Inhibition of SFKs also disrupted H(2)O(2)-mediated chemotaxis of primary human neutrophils. In vitro analysis identified a single cysteine residue, C466, as being responsible for direct oxidation-mediated activation of Lyn. Furthermore, transgenic-tissue-specific reconstitution with wild-type Lyn and a cysteine mutant revealed that Lyn C466 is important for the neutrophil wound response and downstream signalling in vivo. This is the first identification, to our knowledge, of a physiological redox sensor that mediates leukocyte wound attraction in multicellular organisms.

The F-BAR protein PSTPIP1 controls extracellular matrix degradation and filopodia formation in macrophages.

PSTPIP1 is a cytoskeletal adaptor and F-BAR protein that has been implicated in autoinflammatory disease, most notably in the PAPA syndrome: pyogenic sterile arthritis, pyoderma gangrenosum, and acne. However, the mechanism by which PSTPIP1 regulates the actin cytoskeleton and contributes to disease pathogenesis remains elusive. Here, we show that endogenous PSTPIP1 negatively regulates macrophage podosome organization and matrix degradation. We identify a novel PSTPIP1-R405C mutation in a patient presenting with aggressive pyoderma gangrenosum. Identification of this mutation reveals that PSTPIP1 regulates the balance of podosomes and filopodia in macrophages. The PSTPIP1-R405C mutation is in the SRC homology 3 (SH3) domain and impairs Wiskott-Aldrich syndrome protein (WASP) binding, but it does not affect interaction with protein-tyrosine phosphatase (PTP)-PEST. Accordingly, WASP inhibition reverses the elevated F-actin content, filopodia formation, and matrix degradation induced by PSTPIP1-R405C. Our results uncover a novel role for PSTPIP1 and WASP in orchestrating different types of actin-based protrusions. Our findings implicate the cytoskeletal regulatory functions of PSTPIP1 in the pathogenesis of pyoderma gangrenosum and suggest that the cytoskeleton is a rational target for therapeutic intervention in autoinflammatory disease.

Management of advanced ocular surface disease in patients with severe atopic keratoconjunctivitis.

AIM & OBJECTIVE: Severe ocular surface disease, including limbal stem cell deficiency (LSCD) can occur as a consequence of severe atopic keratoconjunctivitis (AKC) that has been inadequately treated. Our goal was to describe the management and outcomes of severe ocular surface disease in AKC patients. METHODS: We performed a retrospective analysis of a case series of 13 eyes of 8 patients with advanced ocular surface disease associated with severe AKC. The clinical presentation, medical and surgical management, and visual and anatomic outcomes were analyzed. RESULTS: Five eyes were treated with medical interventions alone, which included topical or systemic immunomodulatory therapy (IMT) for all eyes. These eyes had a decline in mean visual acuity from LogMAR 0.96 to 2.04 between the initial and final visits related to recurrent epithelial defects or corneal ulceration. Eight eyes were treated with surgical approaches in addition to medical treatment. Initial surgical treatments included limbal stem cell transplantation (n = 5), Boston keratoprosthesis (n = 2), and superficial keratectomy (n = 1). Both eyes that underwent primary keratoprosthesis had severe post-operative complications and became no light perception. In the remainder of the surgically treated eyes, there was an improvement visual acuity from LogMAR 1.43 to 0.6 between the pre-operative and final post-operative visit. CONCLUSION: Visual rehabilitation in eyes severe ocular surface disease due to prolonged AKC is challenging. While some patients did experience improved vision, most eyes did not improve or experienced severe complications with vision loss. Early intervention with immunomodulatory therapy may prevent progression of the disease to advanced stages.

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 reverse migration becomes transparent with zebrafish.

The precise control of neutrophil-mediated inflammation is critical for both host defense and the prevention of immunopathology. In vivo imaging studies in zebrafish, and more recently in mice, have made the novel observation that neutrophils leave a site of inflammation through a process called neutrophil reverse migration. The application of advanced imaging techniques to the genetically tractable, optically transparent zebrafish larvae was critical for these advances. Still, the mechanisms underlying neutrophil reverse migration and its effects on the resolution or priming of immune responses remain unclear. Here, we review the current knowledge of neutrophil reverse migration, its potential roles in host immunity, and the live imaging tools that make zebrafish a valuable model for increasing our knowledge of neutrophil behavior in vivo.

Imaging podosome dynamics and matrix degradation.

Invasive cell migration is critical for leukocyte trafficking into tissues. Podosomes are matrix-degrading adhesive structures that are formed by macrophages and are necessary for macrophage migration and invasion. Here, we describe methods for imaging and quantifying podosomes in primary human macrophages and in THP-1 cells, a monocyte cell line that can be differentiated to a macrophage-like state. Moreover, we outline detailed methods for live imaging of podosomes, which are highly dynamic, and for the quantification of rates of podosome turnover. Finally, we discuss methods for the quantitative analysis of matrix degradation on fluorescent-gelatin-coated cover slips.

Preferential dimethylation of histone H4 lysine 20 by Suv4-20.

Post-translational modifications of histone tails direct nuclear processes including transcription, DNA repair, and chromatin packaging. Lysine 20 of histone H4 is mono-, di-, or trimethylated in vivo, but the regulation and significance of these methylations is poorly understood. The SET domain proteins PR-Set7 and Suv4-20 have been implicated in mono- and trimethylation, respectively; however, enzymes that dimethylate lysine 20 have not been identified. Here we report that Drosophila Suv4-20 is a mixed product specificity methyltransferase that dimethylates approximately 90% and trimethylates less than 5% of total H4 at lysine 20 in S2 cells. Trimethylation, but not dimethylation, is reduced in Drosophila larvae lacking HP1, suggesting that an interaction with HP1 regulates the product specificity of Suv4-20 and enrichment of trimethyllysine 20 within heterochromatin. Similar to the Drosophila enzyme, human Suv4-20h1/h2 enzymes generate di- and trimethyllysine 20. PR-Set7 and Suv4-20 are both required for normal levels of methylation, suggesting they have non-redundant functions. Alterations in the level of lysine 20 methylation following knock-down or overexpression of Suv4-20 did not affect lysine 16 acetylation, revealing that these two modifications are not competitive in vivo. Depletion of Suv4-20h1/h2 in HeLa cells impaired the formation of 53BP1 foci, suggesting dimethyllysine 20 is required for a proper DNA damage response. Collectively, the data indicate that Suv4-20 generates nearly ubiquitous dimethylation that facilitates the DNA damage response and selective trimethylation that is involved in heterochromatin formation.