Microvesicles (MVs) are emerging as a novel means to enact cell-to-cell communication in inflammation. Here, we aimed to ascertain the ability of neutrophil-derived MVs to modulate target cell behaviour, the focus being the macrophage. MVs were generated in response to tumour necrosis factor-α, from healthy control neutrophils or those from rheumatoid arthritis patients. MVs were used to stimulate human monocyte-derived macrophages in vitro, or administered intra-articularly in the K/BxN mouse model of arthritis. A macrophage/fibroblast-like synoviocyte co-culture system was used to study the effects of vesicles on the crosstalk between these cells. We demonstrate a direct role for phosphatidylserine and annexin-A1 exposed by the MVs to counteract classical activation of the macrophages, and promote the release of transforming growth factor-β, respectively. Classically-activated macrophages exposed to neutrophil MVs no longer activated fibroblast-like synoviocytes in subsequent co-culture settings. Finally, intra-articular administration of neutrophil MVs from rheumatoid arthritis patients in arthritic mice affected the phenotype of joint macrophages. Altogether these data, with the identification of specific MV determinants, open new opportunities to modulate on-going inflammation in the synovia - mainly by affecting macrophage polarization and potentially also fibroblast-like synoviocytes - through the delivery of autologous or heterologous MVs produced from neutrophils.

Ischemia/reperfusion (I/R) injury is a severe inflammatory insult associated with numerous pathologies, such as myocardial infarction, stroke and acute kidney injury. I/R injury is characterized by a rapid influx of activated neutrophils secreting toxic free radical species and degrading enzymes that can irreversibly damage the tissue, thus impairing organ functions. Significant efforts have been invested in identifying therapeutic targets to suppress neutrophil recruitment and activation post-I/R injury. In this context, pharmacological targeting of neutrophil elastase (NE) has shown promising anti-inflammatory efficacy in a number of experimental and clinical settings of I/R injury and is considered a plausible clinical strategy for organ care. However, the mechanisms of action of NE, and hence its inhibitors, in this process are not fully understood. Here we conducted a comprehensive analysis of the impact of NE genetic deletion on neutrophil infiltration in four murine models of I/R injury as induced in the heart, kidneys, intestine and cremaster muscle. In all models, neutrophil migration into ischemic regions was significantly suppressed in NE-/- mice as compared with wild-type controls. Analysis of inflamed cremaster muscle and mesenteric microvessels by intravital and confocal microscopy revealed a selective entrapment of neutrophils within venular walls, most notably at the level of the venular basement membrane (BM) following NE deletion/pharmacological blockade. This effect was associated with the suppression of NE-mediated remodeling of the low matrix protein expressing regions within the venular BM used by transmigrating neutrophils as exit portals. Furthermore, whilst NE deficiency led to reduced neutrophil activation and vascular leakage, levels of monocytes and prohealing M2 macrophages were reduced in tissues of NE-/- mice subjected to I/R. Collectively our results identify a vital and non-redundant role for NE in supporting neutrophil breaching of the venular BM post-I/R injury but also suggest a protective role for NE in promoting tissue repair. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B4 as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions.

Neutrophil activation and adhesion must be tightly controlled to prevent complications associated with excessive inflammatory responses. The role of the anti-inflammatory peptide chemerin15 (C15) and the receptor ChemR23 in neutrophil physiology is unknown. Here, we report that ChemR23 is expressed in neutrophil granules and rapidly upregulated upon neutrophil activation. C15 inhibits integrin activation and clustering, reducing neutrophil adhesion and chemotaxis in vitro. In the inflamed microvasculature, C15 rapidly modulates neutrophil physiology inducing adherent cell detachment from the inflamed endothelium, while reducing neutrophil recruitment and heart damage in a murine myocardial infarction model. These effects are mediated through ChemR23. We identify the C15/ChemR23 pathway as a new regulator and thus therapeutic target in neutrophil-driven pathologies.

We have reported the existence of a distinct neutrophil phenotype in giant cell arteritis (GCA) patients arising at week 24 of steroid treatment. In the present study, we investigated whether longitudinal analysis of neutrophil phenotype in patients with polymyalgia rheumatica (PMR) could reveal a novel association with disease status and immune cell cross-talk. Thus, we monitored PMR patient neutrophil phenotype and plasma microvesicle (MV) profiles in blood aliquots collected pre-steroid, and then at weeks 1, 4, 12 and 24 post-steroid treatment.Using flow cytometric and flow chamber analyses, we identified 12-week post-steroid as a pivotal time-point for a marked degree of neutrophil activation, correlating with disease activity. Analyses of plasma MVs indicated elevated AnxA1+ neutrophil-derived vesicles which, in vitro, modulated T-cell reactivity, suggesting distinct neutrophil phenotypic and cross-talk changes at 24 weeks, but not at 12-week post-steroid.Together, these data indicate a clear distinction from GCA patient neutrophil and MV signatures, and provide an opportunity for further investigations on how to 'stratify' PMR patients and monitor their clinical responses through novel use of blood biomarkers.

Inflammatory bowel disease is characterised by intricate immune cell interactions with tissue cells and such cross-talks can become deregulated. The formyl peptide receptor 1 (Fpr1) is expressed by both immune and stromal cells including epithelial cells. We evaluated the development of the physiopathology of the DNBS induced colitis in Fpr1 KO mice on the C57BL/6 genetic background compared to C57BL/6 genetic background animals. We have assessed both macroscopic and histological markers of the diseased, together with the immunohistochemical and molecular changes. DNBS-treated Fpr1 KO mice showed a i) reduction in weight loss, ii) lower extent of colon injury and iii) an increase in MPO activity. Molecular analyses indicated that in absence of Fpr1 there was reduced NF-κB translocation into the nucleus, cytokines levels, FOXP3 and GATA3, CD4, CD8 and CD45 expression as well as a dysregulation of TGF-β signalling. In addition, the colon of DNBS-injected Fpr1 KO mice displayed a lower degree of expression of Bax and higher expression of Bcl-2 compared correspondent WT mice. Finally, intravital microscopy investigation of the microcirculation post-DNBS instillation revealed a lower degree of neutrophil-endothelial cell rolling and adhesion - mediated by P-selectin and ICAM-1 - in Fpr1 KO mice. All the main outcome in the study have a P-value, statistical significance of evidence, less than 0.05. We provide evidence for an important pathogenic role of mouse Fpr1 in experimental colitis, an outcome effected through modulation of immune cell recruitment together with a modulation of local cellular activation and survival.

Annexin A1 (AnxA1) modulates neutrophil life span and bone marrow/blood cell trafficking thorough activation of formyl-peptide receptors (FPRs). Here, we investigated the effect of exogenous AnxA1 on haematopoiesis in the mouse. Treatment of C57BL/6 mice with recombinant AnxA1 (rAnxA1) reduced the granulocyte-macrophage progenitor (GMP) population in the bone marrow, enhanced the number of mature granulocytes Gr-1+Mac-1+ in the bone marrow as well as peripheral granulocytic neutrophils and increased expression of mitotic cyclin B1 on hematopoietic stem cells (HSCs)/progenitor cells (Lin-Sca-1+c-Kit+: LSK). These effects were abolished by simultaneous treatment with Boc-2, an FPR pan-antagonist. In in vitro studies, rAnxA1 reduced both HSC (LSKCD90lowFLK-2-) and GMP populations while enhancing mature cells (Gr1+Mac1+). Moreover, rAnxA1 induced LSK cell proliferation (Ki67+), increasing the percentage of cells in the S/G2/M cell cycle phases and reducing Notch-1 expression. Simultaneous treatment with WRW4, a selective FPR2 antagonist, reversed the in vitro effects elicited by rAnxA1. Treatment of LSK cells with rAnxA1 led to phosphorylation of PCLγ2, PKC, RAS, MEK, and ERK1/2 with increased expression of NFAT2. In long-term bone marrow cultures, rAnxA1 did not alter the percentage of LSK cells but enhanced the Gr-1+Mac-1+ population; treatment with a PLC (U73122), but not with a PKC (GF109203), inhibitor reduced rAnxA1-induced phosphorylation of ERK1/2 and Elk1. Therefore, we identify here rAnxA1 as an inducer of HSC/progenitor cell differentiation, favouring differentiation of the myeloid/granulocytic lineage, via Ca2+/MAPK signalling transduction pathways.