Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology.

Genes located on chromosome 21, over-expressed in Down syndrome (DS) and Alzheimer's disease (AD) and which regulate vesicle trafficking, are strong candidates for involvement in AD neuropathology. Regulator of calcineurin activity 1 (RCAN1) is one such gene. We have generated mutant mice in which RCAN1 is either over-expressed (RCAN1(ox)) or ablated (Rcan1-/-) and examined whether exocytosis from chromaffin cells, a classic cellular model of neuronal exocytosis, is altered using carbon fibre amperometry. We find that Rcan1 regulates the number of vesicles undergoing exocytosis and the speed at which the vesicle fusion pore opens and closes. Cells from both Rcan1-/- and RCAN1(ox) mice display reduced levels of exocytosis. Changes in single-vesicle fusion kinetics are also evident resulting in the less catecholamine released per vesicle with increasing Rcan1 expression. Acute calcineurin inhibition did not replicate the effect of RCAN1 overexpression. These changes are not due to alterations in Ca2+ entry or the readily releasable vesicle pool size. Thus, we illustrate a novel regulator of vesicle exocytosis, Rcan1, which influences both exocytotic rate and vesicle fusion kinetics. If Rcan1 functions similarly in neurons then overexpression of this protein, as occurs in DS and AD brains, will reduce both the number of synaptic vesicles undergoing exocytosis and the amount of neurotransmitter released per fusion event. This has direct implications for the pathogenesis of these diseases as sufficient levels of neurotransmission are required for synaptic maintenance and the prevention of neurodegeneration and vesicle trafficking defects are the earliest hallmark of AD neuropathology.

Ceftazidime-avibactam is highly efficacious against extensive- and multidrug-resistant strains of Mycobacterium tuberculosis.

People with Down syndrome (DS) exhibit abnormal brain structure. Alterations affecting neurotransmission and signalling pathways that govern brain function are also evident. A large number of genes are simultaneously expressed at abnormal levels in DS; therefore, it is a challenge to determine which gene(s) contribute to specific abnormalities, and then identify the key molecular pathways involved. We generated RCAN1-TG mice to study the consequences of RCAN1 over-expression and investigate the contribution of RCAN1 to the brain phenotype of DS. RCAN1-TG mice exhibit structural brain abnormalities in those areas affected in DS. The volume and number of neurons within the hippocampus is reduced and this correlates with a defect in adult neurogenesis. The density of dendritic spines on RCAN1-TG hippocampal pyramidal neurons is also reduced. Deficits in hippocampal-dependent learning and short- and long-term memory are accompanied by a failure to maintain long-term potentiation (LTP) in hippocampal slices. In response to LTP induction, we observed diminished calcium transients and decreased phosphorylation of CaMKII and ERK1/2-proteins that are essential for the maintenance of LTP and formation of memory. Our data strongly suggest that RCAN1 plays an important role in normal brain development and function and its up-regulation likely contributes to the neural deficits associated with DS.

Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3

Neutrophils are critical in the defense against bacterial and fungal pathogens, and they also modulate the inflammatory process. The areas where neutrophils are studied have expanded from the restricted field of antibacterial defense to the modulation of inflammation and finally, to fine-tuning immune responses. As a result, recent studies have shown that neutrophils are implicated in several systemic autoimmune diseases, although exactly how neutrophils contribute to these diseases and the molecular mechanisms responsible are still under investigation. In a group of autoimmune vasculitides associated with anti-neutrophil cytoplasmic antibodies (AAVs), granulomatosis with polyangiitis (GPA) illustrates the concept that autoimmunity can develop against one specific neutrophil protein, namely, proteinase 3 (PR3), one of the four serine protease homologs contained within azurophilic granules. In this review, we will focus on recent molecular analyses combined with functional studies that provide clear evidence that the pathogenic properties of PR3 are not only a result of its enzymatic activity but also mediated by a particular structural element-the hydrophobic patch-which facilitates associations with various proteins and lipids and permits anchorage into the plasma membrane. Furthermore, these unique structural and functional characteristics of PR3 might be key contributors to the systemic inflammation and to the immune dysregulation observed in GPA.

Acute inflammation is the body's response to infection or injury, characterised by the rapid infiltration of polymorphonuclear neutrophils to the site of injury followed by monocytes, which differentiate locally into macrophages. The latter are essential for the removal of effete neutrophils and provided that the harmful agent is eliminated, removal of neutrophils will lead to the resolution of inflammation. Perturbations in this process result in the persistence of inflammation and close control of pathways associated with resolution are necessary to avoid chronic inflammation, autoimmunity, or both. As our understanding of these processes increase, drugs able to trigger pro-resolution pathways may represent an effective strategy for treating chronic inflammatory diseases.

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