Fibrin gums up the works Plasmin is an abundant plasma protease that cleaves and deactivates the clot-associated protein fibrin. Human deficiencies in plasmin and its inactive proenzyme form, plasminogen (PLG), cause severe inflammation in mucosal tissues such as the mouth and eyes. Silva et al . report that, like humans, mice lacking plasminogen accumulate extravascular fibrin and develop an oral pathology that phenocopies human ligneous periodontitis (see the Perspective by Vicanolo and Hidalgo). The excess fibrin activates neutrophils through the αMβ2 (Mac-1) integrin receptor, which triggers the production of reactive oxygen species and neutrophil extracellular traps. Additionally, certain human polymorphisms in the PLG gene were found to be associated with increased likelihood of developing periodontitis, suggesting that fibrin–neutrophil interactions may be an attractive target for future treatments of this prevalent disease. —STS

Natural product biosynthetic pathways contain a plethora of enzymatic tools to carry out difficult biosynthetic transformations. Here we discover an unusual mononuclear iron-dependent methyltransferase that acts in the initiation steps of apratoxin A biosynthesis (AprA MT1). Fe3+-replete AprA MT1 catalyzes one or two methyl transfer reactions on the substrate malonyl-ACP (acyl carrier protein), whereas Co2+, Fe2+, Mn2+ and Ni2+ support only a single methyl transfer. MT1 homologs exist within the “GNAT” (GCN5-related N-acetyltransferase) loading modules of several modular biosynthetic pathways with propionyl, isobutyryl or pivaloyl starter units. GNAT domains are thought to catalyze decarboxylation of malonyl-CoA and acetyl transfer to a carrier protein. In AprA the GNAT domain lacks both decarboxylation and acyl transfer activity. A crystal structure of the AprA MT1-GNAT didomain with bound Mn2+, malonate and the methyl donor S-adenosylmethionine (SAM) reveals that the malonyl substrate is a bidentate metal ligand, indicating that the metal acts as a Lewis acid to promote methylation of the malonyl α-carbon. The GNAT domain is truncated relative to functional homologs. These results afford an expanded understanding of MT1-GNAT structure and activity, and permit the functional annotation of homologous GNAT loading modules both with and without methyltransferases, additionally revealing their rapid evolutionary adaptation in different biosynthetic contexts.

Efficient migration of macrophages to sites of inflammation requires cell surface–bound plasmin(ogen). Here, we investigated the mechanisms underlying the deficits of plasmin(ogen)-mediated macrophage migration in 2 models: murine thioglycollate-induced peritonitis and in vitro macrophage migration. As previously reported, macrophage migration into the peritoneal cavity of mice in response to thioglycollate was significantly impaired in the absence of plasminogen. Fibrin(ogen) deposition was noted in the peritoneal cavity in response to thioglycollate, with a significant increase in fibrin(ogen) in the plasminogen-deficient mice. Interestingly, macrophage migration was restored in plasminogen-deficient mice by simultaneous imposition of fibrinogen deficiency. Consistent with this in vivo finding, chemotactic migration of cultured macrophages through a fibrin matrix did not occur in the absence of plasminogen. The macrophage requirement for plasmin-mediated fibrinolysis, both in vivo and in vitro, was negated by deletion of the major myeloid integrin αMβ2-binding motif on the γ chain of fibrin(ogen). The study identifies a critical role of fibrinolysis in macrophage migration, presumably through the alleviation of migratory constraints imposed by the interaction of leukocytes with fibrin(ogen) through the integrin αMβ2 receptor.

Highlights d GNAT superfamily catalyzes decarboxylation in addition to acyltransfer d A GNAT-like enzyme initiates biosynthesis of some polyketide natural products d Polyketide synthase GNAT-like enzymes catalyze only decarboxylation

The membrane-anchored matrix metalloprotease MT1-MMP is a potent collagenolytic enzyme with a well-established role in extracellular matrix turnover and cellular invasion into collagen-rich tissues. MT1-MMP is highly expressed in various types of cancer and has been demonstrated to be directly involved in several stages of tumor progression, including primary tumor growth, angiogenesis, invasion and metastasis. Osteosarcoma is the most common type of primary bone cancer. This disease is characterized by invasive tumor growth, leading to extensive bone destruction, and metastasis to the lungs. The tumor cells in human osteosarcoma display a strong expression of MT1-MMP, but the role of MT1-MMP in osteosarcoma progression is currently unknown. In this study, we investigated the role of MT1-MMP during various stages of osteosarcoma development. We utilized an optimized orthotopic murine osteosarcoma model and human osteosarcoma cells in which the MT1-MMP gene was knocked out using CRISPR/Cas9. We observed a strong expression of MT1-MMP in wildtype cells of both primary tumors and lung metastases, but, surprisingly, MT1-MMP deficiency did not affect primary tumor growth, bone degradation or the formation and growth of lung metastases. We therefore propose that, unlike findings reported in other cancers, tumor-expressed MT1-MMP is dispensable for all stages of osteosarcoma progression.

Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we document that the clotting factor fibrin is a critical regulator of neutrophil function at mucosal barriers. We demonstrate that fibrin engages neutrophils through the αMβ2 integrin receptor and activates effector functions, including the production of reactive oxygen species and NET formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis. Indeed, the accumulation of fibrin due to Mendelian genetic defects in plasmin leads to severe oral mucosal immunopathology in mice and humans. Concordantly, genetic polymorphisms in the human PLG gene, encoding plasminogen, are associated with common forms of the oral mucosal disease periodontitis. Our work uncovers fibrin as a critical regulator of neutrophil effector function within the mucosal tissue microenvironment and suggests fibrin-neutrophil engagement as a pathogenic instigator and therapeutic target in common mucosal disease.

Wound cleansing agents are routine in wound care and preoperative preparation.Antiseptic activity intends to prevent contaminating microbes from establishing an infection while also raising concerns of cytotoxicity and delayed wound healing. We evaluated the cytotoxicity of five clinically used wound cleaning agents (saline, povidone iodine, Dove ® and Dial ® soaps, and chlorhexidine gluconate [CHG]) using both an ex vivo and in vivo human skin xenograft mouse model, in contrast to classical in vitro models that lack the structural and compositional heterogeneity of human skin. We further established an ex vivo wound contamination model inoculated with $100 cells of Pseudomonas aeruginosa or Staphylococcus aureus to evaluate antimicrobial efficacy. Scanning electron microscopy and confocal microscopy were used to evaluate phenotypic and spatial characteristics of bacterial cells in wound tissue.CHG significantly reduced metabolic activity of the skin explants, while all treatments except saline affected local cellular viability. CHG cytotoxicity persisted and progressed over 14 days, impairing wound healing in vivo. Within the contamination model, CHG treatment resulted in a significant reduction of P. aeruginosa wound surface counts at 24 h post-treatment. However, this effect was transient and serial application of CHG had no effect on both P. aeruginosa or S. aureus microbial growth.Microscopy revealed that viable cells of P. aeruginosa reside deep within wound tissue post-CHG application, likely serving as a reservoir to re-populate the tissue to a high bioburden. We reveal concerning cytotoxicity and limited antimicrobial activity of CHG in human skin using clinically relevant models, with the ability to resolve spatial localization and temporal dynamics of tissue viability and microbial growth.

Introduction Chlorhexidine (CHG) is ubiquitous in surgical perioperative care. In vivo studies of CHG cytotoxicity on human skin are lacking. Given the use of CHG for daily wound cares and as a presurgical scrub, including donor site preparation, we sought to identify if CHG cytotoxicity would persist in a clinically relevant in vivo human skin xenograft model. Methods Human skin tissues were obtained from elective surgeries. Partial thickness wounds were created ex vivo in human skin using a 4 mm punch biopsy. 2% CHG (treatment) or PBS (control) was applied to the wounds for 30 minutes followed by rinsing the tissue +/- mechanical disruptive irrigation. Tissues were cultured at the air-liquid interface for 24 hours in culture media after treatment and tissue viability was performed using an MTT assay. For in vivo studies, athymic mice (n=4) were grafted on bilateral flanks with human skin. Eight weeks after engraftment and normalization of skin architecture, 4 mm partial thickness wounds were created on each xenograft (2 per mouse – treatment and control). 2% CHG was applied daily for 2 minutes followed by irrigation with PBS in the treatment wound. The control wound received PBS application and irrigation. The xenografts received treatment daily for 14 days to mimic daily wound cares, and digital images were obtained to document presence of infection and gross wound healing. On day 14, the xenografts were harvested and stained for lactate dehydrogenase and H&E to assess cell viability and wound re-epithelization, respectively. Results An MTT assay on ex vivo human skin wounds showed that CHG treated groups (irrigation or non-irrigation) had lower cell viability compared to the PBS treated group, however irrigation mitigates the cytotoxicity of CHG on human skin. In the in vivo xenograft study, no signs of infection were identified in either PBS or CHG treated wounds throughout the study. The wound size appeared larger on gross inspection in the CHG treated group compared to the PBS group as early as day 2.Microscopically, the PBS treated wounds were fully re-epithelialized (n=2) or had significantly more re-epithelialization (n=2) than the CHG treated wounds (n=4) after 14 days of treatment. The PBS-treated wounds were viable throughout the tissue, indicating the irrigation procedure was not harmful to the cells. In the CHG-treated wounds, nonviable cells were observed in the dermis beneath the wound that was directly in contact with CHG suggesting penetration of CHG contributes to cytotoxicity in acute wounds. Conclusions Daily CHG use is cytotoxic to human skin and impedes wound healing.