Defective endomembrane dynamics in Rab27a deficiency impairs nucleic acid sensing and cytokine secretion in immune cells.

Endosomal Toll-like receptors (eTLRs) are essential for the sensing of non-self through RNA and DNA detection. Here, using spatiotemporal analysis of vesicular dynamics, super-resolution microscopy studies, and functional assays, we show that endomembrane defects associated with the deficiency of the small GTPase Rab27a cause delayed eTLR ligand recognition, defective early signaling, and impaired cytokine secretion. Rab27a-deficient neutrophils show retention of eTLRs in amphisomes and impaired ligand internalization. Extracellular signal-regulated kinase (ERK) signaling and ß2-integrin upregulation, early responses to TLR7 and TLR9 ligands, are defective in Rab27a deficiency. CpG-stimulated Rab27a-deficient neutrophils present increased tumor necrosis factor alpha (TNF-α) secretion and decreased secretion of a selected group of mediators, including interleukin (IL)-10. In vivo, CpG-challenged Rab27a-null mice show decreased production of type I interferons (IFNs) and IFN-γ, and the IFN-α secretion defect is confirmed in Rab27a-null plasmacytoid dendritic cells. Our findings have significant implications for immunodeficiency, inflammation, and CpG adjuvant vaccination.

Periostin regulates lysyl oxidase through ERK1/2 MAPK-dependent serum response factor in activated cardiac fibroblasts.

Collagen crosslinking, mediated by lysyl oxidase, is an adaptive mechanism of the cardiac repair process initiated by cardiac fibroblasts postmyocardial injury. However, excessive crosslinking leads to cardiac wall stiffening, which impairs the contractile properties of the left ventricle and leads to heart failure. In this study, we investigated the role of periostin, a matricellular protein, in the regulation of lysyl oxidase in cardiac fibroblasts in response to angiotensin II and TGFß1. Our results indicated that periostin silencing abolished the angiotensin II and TGFß1-mediated upregulation of lysyl oxidase. Furthermore, the attenuation of periostin expression resulted in a notable reduction in the activity of lysyl oxidase. Downstream of periostin, ERK1/2 MAPK signaling was found to be activated, which in turn transcriptionally upregulates the serum response factor to facilitate the enhanced expression of lysyl oxidase. The periostin-lysyl oxidase association was also positively correlated in an in vivo rat model of myocardial infarction. The expression of periostin and lysyl oxidase was upregulated in the collagen-rich fibrotic scar tissue of the left ventricle. Remarkably, echocardiography data showed a reduction in the left ventricular wall movement, ejection fraction, and fractional shortening, indicative of enhanced stiffening of the cardiac wall. These findings shed light on the mechanistic role of periostin in the collagen crosslinking initiated by activated cardiac fibroblasts. Our findings signify periostin as a possible therapeutic target to reduce excessive collagen crosslinking that contributes to the structural remodeling associated with heart failure.

NET-associated citrullinated histones promote LDL aggregation and foam cell formation in vitro.

Neutrophils have been recently identified in the atherosclerotic lesion and they can release neutrophil extracellular trap (NET) under the pro-inflammatory conditions prevailing in the lesion. Citrullinated histones (Cit-histones) are the major type of citrullinated proteins associated with NET release. Since elevated levels of citrullinated proteins have been detected in inflammatory diseases including atherosclerosis, this study analysed the role played by NET and Cit-histones in different atherogenic events in vitro. First, neutrophil recruitment and NET release in the presence of low-density lipoprotein (LDL) and oxidised LDL (Ox-LDL) were analysed by Boyden's chamber method and microscopy respectively. Then, LDL oxidation and LDL aggregation in the presence of NET and Cit-histones were analysed spectroscopically. Foam cell formation in the presence of NET or Cit-histone was studied by both microscopic and spectroscopic methods. While neutrophil recruitment was facilitated by Ox-LDL and not by LDL, the extent of NET release was significantly increased in the presence of both LDL and Ox-LDL. In the presence of NET, LDL oxidation, aggregation and foam cell formation were found to be increased. Cit-histones were found to accelerate LDL aggregation and foam cell formation at higher citrulline levels. Altogether, the results suggest that both NET and NET-associated Cit-histone released at the lesion can play major roles as pro-atherogenic mediators. Inhibiting the action of NET or Cit-histone would, therefore, be beneficial in slowing down atherosclerotic progression.

Calcium depletion at high glucose concentration promotes vesicle-mediated NET release in response to Staphylococcus aureus.

The primary immune response against Staphylococcus aureus is mediated by neutrophils. In response to S. aureus and its proteins, neutrophil shows two different kinds of NETosis, viz. suicidal and vesicular NETosis. Glucose is the major energy source of neutrophils for performing NETosis. However, NETosis was found altered in response to high glucose levels. Growth of S. aureus was also found modulated in response to high glucose and they behave differently at different glucose levels. This work was attempted to study NET release in response to S. aureus cell-free culture supernatant at different glucose concentrations. Freshly isolated neutrophils were treated with different concentrations of glucose along with S. aureus cell-free culture supernatant and were analyzed for neutrophil extracellular trap formation, ROS production, and peptidylarginine deiminase 4 activities. Influence of calcium on NETosis was analyzed using calcium chelator (EDTA) and calcium inhibitor (TMB-8). With increasing glucose levels, NET release in response to S. aureus cell-free culture supernatant was increased. Oxidant level was also increased dose-dependently with increasing concentrations of glucose. At very high glucose concentrations (> 15 mM), vesicular NETosis was predominantly observed. At these glucose concentrations, peptidylarginine deiminase activity was found to be decreased. Furthermore, calcium quenching in the medium facilitated vesicular mode of NET release. In conclusion, calcium depletion occurring at high glucose concentrations can reduce peptidylarginine deiminase 4 activity and can thereby promote the vesicular NET release.

Monocyte clearance of apoptotic neutrophils is unhindered in the presence of NETosis, but proteins of NET trigger ETosis in monocytes.

Resolution of inflammation needs effective and timely removal of dead cells and other toxic products of neutrophils, monocytes, and macrophages. In this study, we evaluated the role of monocytes in the clearance of neutrophil extracellular trap (NET) and apoptotic neutrophils in the inflammation site. For this, monocytes were observed microscopically after exposing them with NETs and/or apoptotic bodies. A subset of monocytes exposed to NETs ejected extracellular traps and this was shown to be mediated by proteins like elastase and citrullinated histones present in NET supernatant. Monocytes showed a preference for the internalisation of the apoptotic body when both NET and apoptotic bodies were present in the medium. The study provides new insight into the role of monocytes in the clearance of NET and apoptotic neutrophils and this information may open up a way in formulating therapeutic strategies for accelerating resolution of inflammation.