Signaling In Neutrophils Research Articles

Hydrogen sulfide limits neutrophil transmigration, inflammation, and oxidative burst in lipopolysaccharide-induced acute lung injury

Transmigration and activation of neutrophils in the lung reflect key steps in the progression of acute lung injury (ALI). It is known that hydrogen sulfide (H2S) can limit neutrophil activation, but the respective mechanisms remain elusive. Here, we aimed to examine the underlying pathways in pulmonary inflammation. In vivo, C57BL/6N mice received the H2S slow releasing compound GYY4137 prior to lipopolysaccharide (LPS) inhalation. LPS challenge led to pulmonary injury, inflammation, and neutrophil transmigration that were inhibited in response to H2S pretreatment. Moreover, H2S reduced mRNA expression of macrophage inflammatory protein-2 (MIP-2) and its receptor in lung tissue, as well as the accumulation of MIP-2 and interleukin-1β in the alveolar space. In vitro, GYY4137 did not exert toxic effects on Hoxb8 neutrophils, but prevented their transmigration through an endothelial barrier in the presence and absence of MIP-2. In addition, the release of MIP-2 and reactive oxygen species from LPS-stimulated Hoxb8 neutrophils were directly inhibited by H2S. Taken together, we provide first evidence that H2S limits lung neutrophil sequestration upon LPS challenge. As proposed underlying mechanisms, H2S prevents neutrophil transmigration through the inflamed endothelium and directly inhibits pro-inflammatory as well as oxidative signalling in neutrophils. Subsequently, H2S pretreatment ameliorates LPS-induced ALI.

Cooperative PSGL-1 and CXCR2 signaling in neutrophils promotes deep vein thrombosis in mice

Inflammation is a major contributor to deep vein thrombosis (DVT). Flow restriction of the inferior vena cava (IVC) in mice induces DVT like that in humans. In this model, P-selectin-dependent adhesion of neutrophils and monocytes leads to release of neutrophil extracellular traps (NETs) and expression of tissue factor. However, it is not known what signals cause myeloid cells to generate these procoagulant effectors. Using ultrasonography and spinning-disk intravital microscopy in genetically engineered mice, we found that engagement of P-selectin glycoprotein ligand-1 (PSGL-1) and the chemokine receptor CXCR2 on rolling neutrophils propagated signals that cooperated to induce β2 integrin-dependent arrest in flow-restricted IVCs. Unlike previous reports, PSGL-1 signaling in neutrophils did not require L-selectin, and it used tyrosine 145 rather than tyrosines 112 and 128 on the adaptor Src homology domain-containing leukocyte phosphoprotein of 76 kDa. PSGL-1 and CXCR2 signaling cooperated to increase the frequency and size of thrombi, in part by stimulating release of NETs. Unlike in neutrophils, blocking PSGL-1 or CXCR2 signaling in monocytes did not affect their recruitment into thrombi or their expression of tissue factor. Our results demonstrate that neutrophils cooperatively signal through PSGL-1 and CXCR2 to promote DVT.

Cutting Edge: Dysregulated CARD9 Signaling in Neutrophils Drives Inflammation in a Mouse Model of Neutrophilic Dermatoses.

Mice homozygous for the Y208N amino acid substitution in the carboxy terminus of SHP-1 (referred to as Ptpn6spin mice) spontaneously develop a severe inflammatory disease resembling neutrophilic dermatosis in humans. Disease in Ptpn6spin mice is characterized by persistent footpad swelling and suppurative inflammation. Recently, in addition to IL-1α and IL-1R signaling, we demonstrated a pivotal role for RIPK1, TAK1, and ASK1 in promoting inflammatory disease in Ptpn6spin mice. In the current study we have identified a previously unknown role for CARD9 signaling as a critical regulator for Ptpn6spin-mediated footpad inflammation. Genetic deletion of CARD9 significantly rescued the Ptpn6spin-mediated footpad inflammation. Mechanistically, enhanced IL-1α-mediated signaling in Ptpn6spin mice neutrophils was dampened in Ptpn6spinCard9-/- mice. Collectively, this study identifies SHP-1 and CARD9 cross-talk as a novel regulator of IL-1α-driven inflammation and opens future avenues for finding novel drug targets to treat neutrophilic dermatosis in humans.

The LTB4-BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis.

Neutrophils sense and respond to diverse chemotactic cues through G-protein-coupled receptors (GPCRs). However, the precise trafficking dynamics of chemoattractant GPCRs during neutrophil activation and chemotaxis remain unclear. Here, by using small-molecule inhibitors and CRISPR-based knockouts, we establish that two primary chemoattractant GPCRs - formyl peptide receptor 1 (FPR1) and complement component 5a (C5a) receptor 1 (C5aR1) - internalize in a CDC42-actin-dependent manner. Through live-cell imaging, we demonstrate that, upon stimulation, FPR1 rapidly clusters and re-distributes along the plasma membrane to the trailing edge, where it internalizes and is directionally trafficked towards the front of migrating primary human neutrophils. In contrast to FPR1 and C5aR1, the leukotriene B4 (LTB4) receptor (BLT1, also known as LTB4R), which relays LTB4 signals in response to primary chemoattractants during neutrophil chemotaxis, fails to internalize upon physiological stimulation with LTB4, N-formyl-Met-Leu-Phe (fMLF) or C5a. Importantly, we report that blocking the LTB4-BLT1 axis or downstream myosin activation enhances the internalization of FPR1 and C5aR1, thus reducing downstream signaling and impairing chemotaxis to primary chemoattractants. The polarized trafficking of chemoattractant GPCRs and its regulation by the BLT1-mediated myosin activation therefore drives persistent chemotactic signaling in neutrophils.This article has an associated First Person interview with the first author of the paper.

HoxB8 neutrophils replicate Fcγ receptor and integrin-induced neutrophil signaling and functions.

Neutrophils are short‐lived, terminally differentiated leukocytes that form an essential part of host immunity and play a key role in acute and chronic inflammation. The analysis of these important cells is hindered by the fact that neutrophils are not amenable to culture, transfection, or transduction. Conditionally HoxB8‐immortalized mouse hematopoietic progenitors are suitable for in vitro differentiation of a range of myeloid cells, including neutrophils. Integrins and FcγRs are cell surface receptors, the ligation of which is required for a range of neutrophil functions that are important in health and disease. We show here that HoxB8 neutrophils express major neutrophil integrins and FcγRs. They respond to FcγR and integrin stimulation in a manner that is comparable with primary neutrophils, in terms of intracellular signaling. HoxB8 neutrophils also perform a range of FcγR/integrin‐dependent neutrophil functions, including, generation of reactive oxygen species, degranulation, and chemotaxis. Our findings suggest that HoxB8 neutrophils represent a faithful experimental model system for the analysis of Fc and integrin receptor‐dependent neutrophil functions.

Factor XII in inflammation and wound healing.

This review describes the contribution of coagulation factor XII (FXII) in sterile inflammation and wound healing, focusing on recently identified roles for zymogen FXII in neutrophil functions. Recent studies have identified an important role for FXII in neutrophil trafficking. In particular, following neutrophil activation, autocrine FXII signals through the urokinase plasminogen activator receptor (uPAR) on the neutrophil surface to upregulate neutrophil functions. The sum of these activities leads to neutrophil adhesion, chemotaxis, and neutrophil extracellular (NET) formation. Downregulating FXII-mediated signaling in neutrophils is associated with improved wound healing. These recent findings show the sophisticated role of FXII in vivo and create new opportunities for research on the treatment of chronic inflammatory diseases.

LXR/RXR signaling and neutrophil phenotype following myocardial infarction classify sex differences in remodeling

Sex differences in heart failure development following myocardial infarction (MI) are not fully understood. We hypothesized that differential MI signaling could explain variations in outcomes. Analysis of the mouse heart attack research tool 1.0 (422 mice; young = 5.4 ± 0.1; old = 23.3 ± 0.1 months of age) was used to dissect MI signaling pathways, which was validated in a new cohort of mice (4.8 ± 0.2 months of age); and substantiated in humans. Plasma collected at visit 2 from the MI subset of the Jackson Heart Study (JHS; a community-based study consisting of middle aged and older adults of African ancestry) underwent glycoproteomics grouped by outcome: (1) heart failure hospitalization after visit 2 (n = 3 men/12 women) and (2) without hospitalization through 2012 (n = 24 men/21 women). Compared to young male mice, the infarct region of young females had fewer, but more efficient tissue clearing neutrophils with reduced pro-inflammatory gene expression. Apolipoprotein (Apo) F, which acts upstream of the liver X receptors/retinoid X receptor (LXR/RXR) pathway, was elevated in the day 7 infarcts of old mice compared to young controls and was increased in both men and women with heart failure. In vitro, Apo F stimulated CD36 and peroxisome proliferator-activated receptor (PPAR)γ activation in male neutrophils to turn off NF-κB activation and stimulate LXR/RXR signaling to initiate resolution. Female neutrophils were desensitized to Apo F and instead relied on thrombospondin-1 stimulation of CD36 to upregulate AMP-activated protein kinase, resulting in an overall better wound healing strategy. With age, female mice were desensitized to LXR/RXR signaling, resulting in enhanced interleukin-6 activation, a finding replicated in the JHS community cohort. This is the first report to uncover sex differences in post-MI neutrophil signaling that yielded better outcomes in young females and worse outcomes with age.

Real-time visualisation and quantification of Neutrophil Extracellular Traps

Abstract The ability of neutrophils to release extracellular traps (NETs) is one of several mechanisms by which the body defends against infection. When neutrophils encounter invading pathogens, the cells release a mixture of antimicrobial proteins and chromatin to trap and degrade microbes. NETs have been implicated in a number of disorders including atherosclerosis, systemic lupus erythematosus (SLE) and thrombosis. NETosis can be stimulated in vitro using a number of methods including chemical compounds, microbes and microcrystals. In order to better understand the signaling pathways involved, we developed a fully kinetic live-cell imaging assay for NETosis (96-well format, IncuCyte S3). Using a fluorescent cell impermeant DNA-binding reagent (IncuCyte CytoTox Green), NET release was visualised and quantified in real time. In both primary human neutrophils and differentiated ‘neutrophil-like’ dHL60 cells, PMA (100nM) induced rapid (2h onset, peak 4–6h), time-dependent increase in fluorescence and nuclear degradation. A concomitant increase in myeloperoxidase, elastase (immunofluorescence) and cell-free DNA (Picogreen) was observed, validating the NETosis signal. PMA-induced NETosis was ROS-dependent (CellRox Red) but did not cause externalisation of phosphatidylserine (PS, Annexin V). In contrast, ionomycin (5mM) induced NETosis more rapidly (onset time <15′), did not induce ROS but did externalise PS. Neutrophils and dHL60 cells were both able to phagocytose bacterial bioparticles (IncuCyte pHrodo-E-coli). Taken together, these data illustrate how NETs and neutrophil signaling pathways can be robustly, meaningfully and efficiently analysed using automated live-cell imaging and compatible detection reagents.

Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils

AbstractRolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)–dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins.

Deficiency of FAM3D (Family With Sequence Similarity 3, Member D), A Novel Chemokine, Attenuates Neutrophil Recruitment and Ameliorates Abdominal Aortic Aneurysm Development.

Chemokine-mediated neutrophil recruitment contributes to the pathogenesis of abdominal aortic aneurysm (AAA) and may serve as a promising therapeutic target. FAM3D (family with sequence similarity 3, member D) is a recently identified novel chemokine. Here, we aimed to explore the role of FAM3D in neutrophil recruitment and AAA development. FAM3D was markedly upregulated in human AAA tissues, as well as both elastase- and CaPO4-induced mouse aneurysmal aortas. FAM3D deficiency significantly attenuated the development of AAA in both mouse models. Flow cytometry analysis indicated that FAM3D-/- mice exhibited decreased neutrophil infiltration in the aorta during the early stage of AAA formation compared with their wild-type littermates. Moreover, application of FAM3D-neutralizing antibody 6D7 through intraperitoneal injection markedly ameliorated elastase-induced AAA formation and neutrophil infiltration. Further, in vitro coculture experiments with FAM3D-neutralizing antibody 6D7 and in vivo intravital microscopic analysis indicated that endothelial cell-derived FAM3D induced neutrophil recruitment. Mechanistically, FAM3D upregulated and activated Mac-1 (macrophage-1 antigen) in neutrophils, whereas inhibition of FPR1 (formyl peptide receptor 1) or FPR2 significantly blocked FAM3D-induced Mac-1 activation, indicating that the effect of FAM3D was dependent on both FPRs. Moreover, specific inhibitors of FPR signaling related to Gi protein or β-arrestin inhibited FAM3D-activated Mac-1 in vitro, whereas FAM3D deficiency decreased the activation of both FPR-Gi protein and β-arrestin signaling in neutrophils in vivo. FAM3D, as a dual agonist of FPR1 and FPR2, induced Mac-1-mediated neutrophil recruitment and aggravated AAA development through FPR-related Gi protein and β-arrestin signaling.

Metabololipidomic profiling of functional immunoresolvent clusters and eicosanoids in mammalian tissues

Metabolomics enables a systems approach to interrogate the bioactive mediators, their pathways and further metabolites involved in the physiology and pathophysiology of human and animal tissues. New metabololipidomic approaches with mass spectrometry presented in this brief review can now be utilized for the identification and profiling of lipid mediator networks that control inflammation-resolution in human blood and healthy and diseased solid tissues. Coagulation of blood is a protective response that prevents excessive bleeding on injury of blood vessels. Here, we review novel approaches to understand the relationship(s) between coagulation and resolution of inflammation and infection. To determine whether coagulation is involved in host-protective actions by lipid mediators, we used a metabololipidomic-based profiling approach with human whole blood (WB) during coagulation. We identified recently temporal clusters of endogenously produced pro-thrombotic and proinflammatory lipid mediators (eicosanoids), as well as specialized proresolving mediators (SPMs) in this vital process. In addition to the classic eicosanoids (prostaglandins, thromboxanes and leukotrienes), a specific SPM cluster was identified that consists of resolvin E1 (RvE1), RvD1, RvD5, lipoxin B4, and maresin 1, each of which present at bioactive concentrations (0.1–1 nM). The removal of adenosine from coagulating blood samples significantly enhances SPM amounts and unleashes the biosynthesis of RvD3, RvD4, and RvD6 evident following rapid snap freezing with centrifugation before extraction and LC-MS-MS. The classic cyclooxygenase inhibitors, celecoxib and indomethacin, that block thromboxanes and prostanoids do not block production of the clot-driven SPM cluster. Unbiased mass cytometry analysis demonstrated that the SPM cluster produced in human blood targets leukocytes at the single-cell level, directly activating extracellular signaling in human neutrophils and monocytes. Human whole blood treated with the components of this SPM cluster enhanced both phagocytosis and killing of Escherichia coli by leukocytes. Thus, we identified a pro-resolving lipid mediator circuit and specific SPM cluster that promotes host defense. This new lipid mediator (LM)-SPM metabololipidomic approach now provides accessible metabolomic profiles in healthy and diseased human tissues, including cancer, for precision and personalized medicine.

Factor XII and uPAR upregulate neutrophil functions to influence wound healing.

Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12-/-) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor-mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMβ2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12-/- mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12-/- hosts was sufficient to correct the neutrophil migration defect in F12-/- mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.

Identification of Electrophysiological Changes in Alzheimer's Disease: A Microarray Based Transcriptomics and Molecular Pathway Analysis Study

Involvement of amyloid beta and tau proteins in pathogenesis of Alzheimer's disease (AD) has been studied extensively. However, electrophysiological activity, and cellular processes like membrane transport are mostly unstudied. Electrophysiological processes provide a bridge between brain activity and cognition, and show promise as translatable biomarkers in preclinical and clinical applications. Biochemical imbalance leads to change in glutamate-based neurotransmission, antioxidant capacity, and in membrane polarization-repolarization events, eventually, resulting in AD. We hypothesize that in AD, these processes are unified at a single metabolic hub and we carried out a holistic system-biology approach. In the present study, we integrated and analyzed multiple AD expression datasets from the GEO database to identify significant genes associated with electrophysiological pathways and attempted determination of interconnected canonical molecular pathways. Partek Genomic suite based expression analysis identified 200 significantly expressed genes using cut-off value of ≤ 0.05 and 2 fold change. Transducer of ERBB2, 2 (TOB2); lactotransferrin (LFT) and RAS-like, family 12 (RASL12) were most up-regulated genes, while neurofilament light polypeptide (NEFL); collagen, type V, alpha 2 (COL5A2); visinin-like 1 (VSNL1); cannabinoid receptor 1 (brain) (CNR1); neurofilament, medium polypeptide (NEFM); regulator of G-protein signaling 4 (RGS4), and synaptosomalassociated protein, 25kDa (SNAP25) were most down-regulated ones. Interestingly, we found majority of transporter genes identified in dataset as downregulated. Ingenuity pathways analysis revealed glutamate receptor signaling, CREB signaling, dopamine- DARPP32 feedback in cAMP signaling, fMLP signaling in neutrophils, and synaptic long term potentiation pathway playing critical role in AD pathophysiology and having correlation with electrophysiological dysfunction.

Predicting pathway cross-talks in ankylosing spondylitis through investigating the interactions among pathways.

Given that the pathogenesis of ankylosing spondylitis (AS) remains unclear, the aim of this study was to detect the potentially functional pathway cross-talk in AS to further reveal the pathogenesis of this disease. Using microarray profile of AS and biological pathways as study objects, Monte Carlo cross-validation method was used to identify the significant pathway cross-talks. In the process of Monte Carlo cross-validation, all steps were iterated 50 times. For each run, detection of differentially expressed genes (DEGs) between two groups was conducted. The extraction of the potential disrupted pathways enriched by DEGs was then implemented. Subsequently, we established a discriminating score (DS) for each pathway pair according to the distribution of gene expression levels. After that, we utilized random forest (RF) classification model to screen out the top 10 paired pathways with the highest area under the curve (AUCs), which was computed using 10-fold cross-validation approach. After 50 bootstrap, the best pairs of pathways were identified. According to their AUC values, the pair of pathways, antigen presentation pathway and fMLP signaling in neutrophils, achieved the best AUC value of 1.000, which indicated that this pathway cross-talk could distinguish AS patients from normal subjects. Moreover, the paired pathways of SAPK/JNK signaling and mitochondrial dysfunction were involved in 5 bootstraps. Two paired pathways (antigen presentation pathway and fMLP signaling in neutrophil, as well as SAPK/JNK signaling and mitochondrial dysfunction) can accurately distinguish AS and control samples. These paired pathways may be helpful to identify patients with AS for early intervention.

What We're Reading

Multiple checkpoints (from U.S. General Accounting Office via Flickr)Checkpoint blockade has been a successful strategy in the unleashing of specific T-cell antitumor responses. The inhibitory receptor IL1R8 is highly expressed on human NK cells and increases during NK-cell maturation and activation. Through the use of IL1R8-deficient mice, IL1R8 was found to act as a checkpoint to NK cell–mediated antitumor and antiviral protection, suggesting that its blockade during therapy could enhance NK-cell activity.Molgora M, …, Mantovani A. Nature 2017 Oct 25. doi:10.1038/nature24293.MHC on DCs (from P Müller et al. Cancer Immunol Res 2014)Which peptides are presented to T cells depends on available HLA alleles. Marty et al. found that find that recurrent tumor mutations generally contain poorly presented peptides, and if not well presented by a patient's HLA, then the probability increases that such mutations will appear in progressing tumors. McGranahan et al. developed a computational tool, LOHHLA, that uses sequencing data to measure the loss of HLA alleles in tumors. Allele loss was common in non–small cell lung cancer with high neoepitope loads and provided a mechanism for immune escape and outgrowth of mutated subclones.Marty R, …, Carter H. Cell 2017 Oct 26. DOI: 10.1016/j.cell.2017.09.050.McGranahan N, …, Swanton C. Cell 2017 Oct 26. DOI: 10.1016/j.cell.2017.10.001.Amphiregulin (from the RCSB PDB)Amphiregulin (Areg), a ligand for the epidermal growth factor receptor, was found to be produced by intratumoral regulatory and conventional CD4+ T cells. Areg was a positive factor in tumor growth, but a T cell–restricted deficiency of Areg did not impact antitumor immunity. Areg's effects were likely indirect, working through tumor-supportive stromal tissue.Green JA, …, Rudensky AY. J Exp Med 2017 Oct 16. DOI: 10.1084/jem.20170356.Neutrophils (from Manuel et al. Cancer Immunol Res 2014)c-MET primarily promotes oncogenesis when expressed on tumor cells, but inhibiting c-MET can also enhance antitumor immunity. This antitumor effect is due to inhibition of c-MET signaling in neutrophils, which prevents their recruitment to, and suppressive influence on, the tumor microenvironment, increasing the efficacy of adoptive T-cell and checkpoint immunotherapies.Glodde N, …, Hölzel M. Immunity 2017 Oct 17;47:789–802.e9.Long noncoding RNA (from Rfam Database via Wikimedia)Viral infection can induce a long noncoding RNA (lncRNA-AC0D1) that, independent of type 1 interferons or the IRF3 pathway, enhances viral replication; eliminating it reduces viral titers. lncRNA-AC0D1 directly binds to GOT2, a key metabolic enzyme that mediates lncRNA-AC0D1 effects. Mutational and functional analyses revealed that lncRNA binding enhanced GOT2 catalytic activity. Intervening with this metabolic pathway in metabolically dysregulated settings such as tumor microenvironments may prove useful.Wang P, …, Cao X. Science Oct 26. DOI: 10.1126/science.aao0409.Unanticipated beneficial outcomes (from N. Potanin via Flickr)A clinical trial with 10,000 atherosclerosis patients (CANTOS) measured the effects of canakinumab, an antibody to IL1β, on cardiac events and also tracked the frequency of subsequent tumors. Those developing lung cancer had higher baseline IL6 and C-reactive protein. Lung, unlike other cancers, arose significantly less frequently if patients received canakinumab, supporting this pathway's importance in lung cancer and highlighting it for potential targeting.Ridker PM, …, Glynn RJ. Lancet 2017 Oct 21;390:1833–42.

Modeling neutrophil migration in dynamic chemoattractant gradients: assessing the role of exosomes during signal relay.

Migrating cells often exhibit signal relay, a process in which cells migrating in response to a chemotactic gradient release a secondary chemoattractant to enhance directional migration. In neutrophils, signal relay toward the primary chemoattractant N--formylmethionyl-leucyl-phenylalanine (fMLP) is mediated by leukotriene B4 (LTB4). Recent evidence suggests that the release of LTB4 from cells occurs through packaging in exosomes. Here we present a mathematical model of neutrophil signal relay that focuses on LTB4 and its exosome-mediated secretion. We describe neutrophil chemotaxis in response to a combination of a defined gradient of fMLP and an evolving gradient of LTB4, generated by cells in response to fMLP. Our model enables us to determine the gradient of LTB4 arising either through directed secretion from cells or through time-varying release from exosomes. We predict that the secondary release of LTB4 increases recruitment range and show that the exosomes provide a time delay mechanism that regulates the development of LTB4 gradients. Additionally, we show that under decaying primary gradients, secondary gradients are more stable when secreted through exosomes as compared with direct secretion. Our chemotactic model, calibrated from observed responses of cells to gradients, thereby provides insight into chemotactic signal relay in neutrophils during inflammation.

The C5a/C5aR1 axis controls the development of experimental allergic asthma independent of LysM-expressing pulmonary immune cells.

C5a regulates the development of maladaptive immune responses in allergic asthma mainly through the activation of C5a receptor 1 (C5aR1). Yet, the cell types and the mechanisms underlying this regulation are ill-defined. Recently, we described increased C5aR1 expression in lung tissue eosinophils but decreased expression in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells (cDCs) and monocyte-derived DCs (moDCs) during the allergic effector phase using a floxed green fluorescent protein (GFP)-C5aR1 knock-in mouse. Here, we determined the role of C5aR1 signaling in neutrophils, moDCs and macrophages for the pulmonary recruitment of such cells and the importance of C5aR1-mediated activation of LysM-expressing cells for the development of allergic asthma. We used LysM-C5aR1 KO mice with a specific deletion of C5aR1 in LysMCre-expressing cells and confirmed the specific deletion of C5aR1 in neutrophils, macrophages and moDCs in the airways and/or the lung tissue. We found that alveolar macrophage numbers were significantly increased in LysM-C5aR1 KO mice. Induction of ovalbumin (OVA)-driven experimental allergic asthma in GFP-C5aR1fl/fl and LysM-C5aR1 KO mice resulted in strong but similar airway resistance, mucus production and Th2/Th17 cytokine production. In contrast, the number of airway but not of pulmonary neutrophils was lower in LysM-C5aR1 KO as compared with GFP-C5aR1fl/fl mice. The recruitment of macrophages, cDCs, moDCs, T cells and type 2 innate lymphoid cells was not altered in LysM-C5aR1 KO mice. Our findings demonstrate that C5aR1 is critical for steady state control of alveolar macrophage numbers and the transition of neutrophils from the lung into the airways in OVA-driven allergic asthma. However, C5aR1 activation of LysM-expressing cells plays a surprisingly minor role in the recruitment and activation of such cells and the development of the allergic phenotype in OVA-driven experimental allergic asthma.

Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88.

Porphyromonas gingivalis is a gram-negative anaerobic periodontal pathogen that persists in dysbiotic mixed-species biofilms alongside a dense inflammatory infiltrate of neutrophils and other leukocytes in the subgingival areas of the periodontium. Toll-like receptor 2 (TLR2) mediates the inflammatory response to P. gingivalis and TLR2-deficient mice resist alveolar bone resorption following oral challenge with this organism. Although, MyD88 is an adaptor protein considered necessary for TLR2-induced inflammation, we now report for the first time that oral challenge with P. gingivalis leads to alveolar bone resorption in the absence of MyD88. Indeed, in contrast to prototypical TLR2 agonists, such as the lipopeptide Pam3CSK4 that activates TLR2 in a strictly MyD88-dependent manner, P. gingivalis strikingly induced TLR2 signaling in neutrophils and macrophages regardless of the presence or absence of MyD88. Moreover, genetic or antibody-mediated inactivation of TLR2 completely reduced cytokine production in P. gingivalis-stimulated neutrophils or macrophages, suggesting that TLR2 plays a non-redundant role in the host response to P. gingivalis. In the absence of MyD88, inflammatory TLR2 signaling in P. gingivalis-stimulated neutrophils or macrophages depended upon PI3K. Intriguingly, TLR2-PI3K signaling was also critical to P. gingivalis evasion of killing by macrophages, since their ability to phagocytose this pathogen was reduced in a TLR2 and PI3K-dependent manner. Moreover, within those cells that did phagocytose bacteria, TLR2-PI3K signaling blocked phago-lysosomal maturation, thereby revealing a novel mechanism whereby P. gingivalis can enhance its intracellular survival. Therefore, P. gingivalis uncouples inflammation from bactericidal activity by substituting TLR2-PI3K in place of TLR2-MyD88 signaling. These findings further support the role of P. gingivalis as a keystone pathogen, which manipulates the host inflammatory response in a way that promotes bone loss but not bacterial clearance. Modulation of these host response factors may lead to novel therapeutic approaches to improve outcomes in disease conditions associated with P. gingivalis.

Reactivation of Gαi-coupled formyl peptide receptors is inhibited by Gαq-selective inhibitors when induced by signals generated by the platelet-activating factor receptor.

Formyl peptide receptor (FPR)-desensitized neutrophils display increased production/release of superoxide (O2-) when activated by platelet-activating factor (PAF), a priming of the response achieved through a unique receptor crosstalk mechanism. The aim of this study was to determine the effect of an inhibitor selective for small, heterotrimeric G proteins belonging to the Gαq subclass on that receptor crosstalk. We show that signals generated by FPRs and the PAF receptor (PAFR) induce activation of the neutrophil O2-, producing NADPH-oxidase, and that response was sensitive to Gαq inhibition in cells activated by PAF, but no inhibition was obtained in cells activated by FPR agonists. Signaling in naive neutrophils is terminated fairly rapidly, and the receptors become homologously desensitized. The downstream sensitivity to Gαq inhibition in desensitized cells displaying increased production/release of O2- through the PAFR receptor crosstalk mechanism also comprised the reactivation of the FPRs, and the activation signals were redirected from the PAFR to the desensitized/reactivated FPRs. The Gαq-dependent activation signals generated by the PAFRs activate the Gαi-coupled FPRs, a receptor crosstalk that represents a novel pathway by which G protein-coupled receptors can be regulated and signaling can be turned on and off.

Applying label-free dynamic mass redistribution assay for studying endogenous FPR1 receptor signalling in human neutrophils

IntroductionThe label-free dynamic mass redistribution-based assay (DMR) is a powerful method for studying signalling pathways of G protein-coupled receptors (GPCRs). Herein we present the label-free DMR assay as a robust readout for pharmacological characterization of formyl peptide receptors (FPRs) in human neutrophils. MethodsNeutrophils were isolated from fresh human blood and their responses to FPR1 and FPR2 agonists, i.e. compound 43, fMLF and WKYMVm were measured in a label-free DMR assay using Epic Benchtop System from Corning®. Obtained DMR traces were used to calculate agonist potencies. ResultsThe potencies (pEC50) of fMLF, WKYMVm and compound 43, determined on human neutrophils using the label-free DMR assay were 8.63, 7.76 and 5.92, respectively. The DMR response to fMLF, but not WKYMVm and compound 43 could be blocked by the FPR1-specific antagonist cyclosporin H. DiscussionWe conclude that the DMR assay can be used, and complements more traditional methods, to study the signalling and pharmacology of endogenous FPR receptors in human neutrophils.