ARAP3 protects from excessive formylated peptide-induced microvascular leakage by acting on endothelial cells and neutrophils.

Vascular permeability is temporarily heightened during inflammation, but excessive inflammation-associated microvascular leakage can be detrimental, as evidenced in the inflamed lung. Formylated peptides regulate vascular leakage indirectly via formylated peptide receptor-1 (FPR1)-mediated recruitment and activation of neutrophils. Here we identify how the GTPase-activating protein ARAP3 protects against formylated peptide-induced microvascular permeability via endothelial cells and neutrophils. In vitro, Arap3-/- endothelial monolayers were characterised by enhanced formylated peptide-induced permeability due to upregulated endothelial FPR1 and enhanced vascular endothelial cadherin internalisation. In vivo, enhanced inflammation-associated microvascular leakage was observed in Arap3-/- mice. Leakage of plasma protein into the lungs of Arap3-/- mice increased within hours of formylated peptide administration. Adoptive transfer experiments indicated this was dependent upon ARAP3 deficiency in both immune and non-immune cells. Bronchoalveolar lavages of formylated peptide-challenged Arap3-/- mice contained neutrophil extracellular traps (NETs). Pharmacological inhibition of NET formation abrogated excessive microvascular leakage, indicating a critical function of NETs in this context. The observation that Arap3-/- mice developed more severe influenza suggests these findings are pertinent to pathological situations characterised by abundant formylated peptides. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

House Dust Mite Allergens and the Induction of Monocyte Interleukin 1ß Production That Triggers an IκBζ-Dependent Granulocyte Macrophage Colony-Stimulating Factor Release from Human Lung Epithelial Cells.

Asthma is a chronic lung disease characterized by inflammation centered upon bronchial epithelium. House dust mite is one of the most common respiratory allergens that trigger exacerbations of asthma. IκBζ (gene NFKBIZ) is a recently recognized member of the NF-κB family that can be induced in mononuclear phagocytes and lung epithelial cells and has been shown to play a prominent role in epithelial cell function. We therefore analyzed the role of IκBζ in regulating lung epithelial cell cytokine responses to house dust mite mix (HDM). We found that human bronchial epithelial cells express IκBζ and release IL-6 and granulocyte macrophage colony-stimulating factor (GMCSF) when cocultured with human monocytes and HDM. This response is blocked in the presence of IL-1 receptor antagonist (IL-1Ra), indicating that it is IL-1 mediated. Neither HDM-stimulated macrophages nor dendritic cells release IL-1ß and subsequently induce cytokine release from the bronchial epithelial cells. Rhodobacter sphaeroides LPS (RS-LPS), a TLR4 antagonist, blocks the ability of HDM to induce IκBζ and release GMCSF from epithelial cells cocultured with monocytes. Additionally, human bronchial epithelial cells show no induction of IκBζ or cytokine responses to direct HDM stimulation. Finally, NFKBIZ small interfering RNA-mediated knockdown in the bronchial epithelial cells suppresses the release of IL-1-induced IL-6 and GMCSF. Our findings indicate a possible role for monocyte recruitment and lung epithelial cell IκBζ in mediating asthma associated inflammation. Thus, IκBζ, IL-1Ra, and RS-LPS deserve future study as potential modulators of house dust mite-induced asthma.

Oxidatively modified low density lipoprotein (LDL) inhibits TLR2 and TLR4 cytokine responses in human monocytes but not in macrophages.

Inflammation characterized by the expression and release of cytokines and chemokines is implicated in the development and progression of atherosclerosis. Oxidatively modified low density lipoproteins, central to the formation of atherosclerotic plaques, have been reported to signal through Toll-like receptors (TLRs), TLR4 and TLR2, in concert with scavenger receptors to regulate the inflammatory microenvironment in atherosclerosis. This study evaluates the role of low density lipoproteins (LDL) and oxidatively modified LDL (oxmLDL) in the expression and release of proinflammatory mediators IκBζ, IL-6, IL-1ß, TNFα, and IL-8 in human monocytes and macrophages. Although standard LDL preparations induced IκBζ along with IL-6 and IL-8 production, this inflammatory effect was eliminated when LDL was isolated under endotoxin-restricted conditions. However, when added with TLR4 and TLR2 ligands, this low endotoxin preparation of oxmLDL suppressed the expression and release of IL-1ß, IL-6, and TNFα but surprisingly spared IL-8 production. The suppressive effect of oxmLDL was specific to monocytes as it did not inhibit LPS-induced proinflammatory cytokines in human macrophages. Thus, TLR ligand contamination of LDL/oxmLDL preparations can complicate interpretations of inflammatory responses to these modified lipoproteins. In contrast to providing a proinflammatory function, oxmLDL suppresses the expression and release of selected proinflammatory mediators.

Immune complex-induced apoptosis and concurrent immune complex clearance are anti-inflammatory neutrophil functions.

Persistent neutrophilic inflammation drives host damage in autoimmune diseases that are characterized by abundant immune complexes. Insoluble immune complexes (iICs) potently activate pro-inflammatory neutrophil effector functions. We and others have shown that iICs also promote resolution of inflammation via stimulation of neutrophil apoptosis. We demonstrate here that iICs trigger FcγRIIa-dependent neutrophil macropinocytosis, leading to the rapid uptake, and subsequent degradation of iICs. We provide evidence that concurrent iIC-induced neutrophil apoptosis is distinct from phagocytosis-induced cell death. First, uptake of iICs occurs by FcγRII-stimulated macropinocytosis, rather than phagocytosis. Second, production of reactive oxygen species, but not iIC-internalization is a pre-requisite for iIC-induced neutrophil apoptosis. Our findings identify a previously unknown mechanism by which neutrophils can remove pro-inflammatory iICs from the circulation. Together iIC clearance and iIC-induced neutrophil apoptosis may act to prevent the potential escalation of neutrophilic inflammation in response to iICs.

IκBζ Regulates Human Monocyte Pro-Inflammatory Responses Induced by Streptococcus pneumoniae.

Pneumococcal lung infections represent a major cause of death worldwide. Single nucleotide polymorphisms (SNPs) in the NFKBIZ gene, encoding the transcription factor IκBζ, are associated with increased susceptibility to invasive pneumococcal disease. We hence analyzed how IκBζ might regulate inflammatory responses to pneumococcal infection. We first demonstrate that IκBζ is expressed in human blood monocytes but not in bronchial epithelial cells, in response to wild type pneumococcal strain D39. D39 transiently induced IκBζ in a dose dependent manner, with subsequent induction of downstream molecules involved in host defense. Of these molecules, IκBζ knockdown reduced the expression of IL-6 and GMCSF. Furthermore, IκBζ overexpression increased the activity of IL-6 and GMCSF promoters, supporting the knockdown findings. Pneumococci lacking either pneumolysin or capsule still induced IκBζ. While inhibition of TLR1/TLR2 blocked D39 induced IκBζ expression, TLR4 inhibition did not. Blockade of p38 MAP kinase and NFκB suppressed D39 induced IκBζ. Overall, our data demonstrates that IκBζ regulates monocyte inflammatory responses to Streptococcus pneumoniae by promoting the production of IL-6 and GMCSF.

Receptor interacting protein-2 plays a critical role in human lung epithelial cells survival in response to Fas-induced cell-death.

Lung epithelial cell death is critical to the lung injury that occurs in the acute respiratory distress syndrome. It is known that FasL plays a prominent role in this lung cell death pathway and may work in part through activation of the receptor interacting protein-2 (RIP2). RIP2 is serine/threonine kinase with a C-terminal caspase activation and recruitment domain (CARD). This CARD contains a highly conserved, predicted tyrosine phosphorylation site. Thus, involvement of tyrosine phosphorylation in the CARD domain of RIP2 may play a critical role in Fas-mediated apoptosis in the human lung immune system. To test this hypothesis, human lung epithelial cells (BEAS-2B) were induced to undergo cell death in response to the Fas agonist antibody CH11 with and without manipulation of endogenous RIP2 concentrations. We show that CH11 increases lung epithelial cell death in a dose-dependent manner as determined by LDH release and nuclear condensation. Fas-induced LDH release was inhibited by RIP2 knock-down. Reduced levels of RIP2 in BEAS-2B cells after treatment with RIP2 siRNA were confirmed by immunoblot. Overexpression of RIP2 in BEAS-2B cells synergized with Fas ligand-induced LDH release in a dose-dependent manner. Finally, mutation of the tyrosine phosphorylation site in CARD of RIP2 protected BEAS-2B cells from Fas ligand induced cell death. Thus RIP2's CARD tyrosine phosphorylation may represent a new therapeutic target to promote the survival of human lung epithelial cells in disorders that lead to acute lung injury and ARDS.