Citrullinated histone 3 causes endothelial barrier dysfunction

Abstract

Circulating components of neutrophil extracellular traps (NETs), especially histones, are associated with tissue injury during inflammatory conditions like sepsis. Commonly used as a NET biomarker, citrullinated histone 3 (H3Cit) may also functionally contribute to the NET-associated inflammatory response. To this end, we sought to examine the role of H3Cit in mediating microvascular endothelial barrier dysfunction. Here we show that H3Cit can directly contribute to inflammatory injury by disrupting the microvascular endothelial barrier. We found that endothelial responses to H3Cit are characterized by cell-cell adherens junction opening and cytoskeleton reorganization with increased F-actin stress fibers. Several signaling pathways often implicated in the transduction of hyperpermeability, such as Rho and MLCK, did not appear to play a major role; however, the adenylyl cyclase activator forskolin blocked the endothelial barrier effect of H3Cit. Taken together, the data suggest that H3Cit-induced endothelial barrier dysfunction may hold promise to treat inflammatory injury.

Citrullinated Histone H3 as a Therapeutic Target for Endotoxic Shock in Mice

“…In this study, the CitH3 peptide was shown to increase permeability of HUVECs in vitro (Figure 1A). In addition, infusion of CitH3 protein has been shown to induce an extravasation of fluorescent-labeled albumin across mouse mesenteric micro vessels without cell death (25). These observations suggest that CitH3 might be one of the causes for vascular leakage and organ edema in sepsis.…”

“…The function of CitH3 remains largely unknown. It was recently reported that human recombinant CitH3 protein could disrupt endothelial barrier in vivo, probably through opening cell-cell adheres junctions and reorganizing the actin cytoskeleton (25). This conclusion needs further evaluation since the recombinant CitH3 protein was catalyzed by PAD4 purified from E. coli, and endotoxin contamination can compromise the validity of the experimental results (26,27).…”

Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7–dependent lupus

“…While there are some similarities in the proteins targeted for citrullination by PAD2 and PAD4, some proteins are selectively targeted, and this could account for the differences in the properties of the NETs. A recent report indicates that Cit-H3 promotes dysfunction of the endothelial barrier through reorganization of the actin cytoskeleton (48), and this may explain why mice lacking Padi4 generate NETs devoid of vasculopathic effects. Future studies in mice that genetically lack both PAD2 and PAD4, as well as in vivo use of PAD2-and PAD4-specific inhibitors, will be required to assess whether dual blockade of these pathways promotes synergistic effects in the modulation of autoimmunity and endothelial dysfunction when compared with inhibition of single isozymes, given the effectiveness previously observed with pan-PAD inhibitors in other lupus mouse models (31,32).…”

“…Neutrophils were isolated from untreated FVB, Padi2 -/-, or Padi4 -/mice and stimulated with imiquimod (R837; 100 μg/ml; InvivoGen) in serum-free RPMI 1640 for 6 hours at 37°C. NETs were isolated as previously described (47) using 10 U/ml of micrococcal nuclease (Thermo Fisher Scientific) for 15 minutes at 37°C (48). NETs were centrifuged to remove cellular debris and the supernatants stored at -80°C until used.…”

Neutrophil extracellular traps in nasal secretions of patients with stable and exacerbated chronic rhinosinusitis and their contribution to induce chemokine secretion and strengthen the epithelial barrier

“…24 Cultured epithelial cells were incubated with (a) NETs recovered upon stimulation of 1 × 10 6 neutrophils (250 ng/mL); (b) with supernatants of neutrophils incubated with NET inhibitor, DPI (10 µL); (c) with human recombinant citrullinated histone 3, a key component to determine the presence of NETs, (H3Cit, 1 µmol/L), (Cayman Chemical); and (d) with supernatants of unstimulated neutrophils (10 µL) for 24 hours, as described previously. 23,25 Thereafter, cultured cells and supernatant were collected to evaluate the expression levels of chemokines with real-time PCR and ELISA. Epithelial permeability and transepithelial electrical resistance were also measured as described previously.…”