Turning chemoattractant receptors on and off with conventional ligands and allosteric modulators: recent advances in formyl peptide receptor signaling and regulation

Recruitment and activation of neutrophils at sites of infection/inflammation relies largely on the surface expression of G-protein coupled receptors (GPCRs) that recognize chemoattractants. One of these receptors, FPR1, for which formylated peptides generated by bacteria and mitochondria are high affinity agonists, was among the first human neutrophil GPCR to being cloned. This receptor shares large sequence homologies with FPR2, another member of the FPR-family expressed in human neutrophils and having a distinct ligand binding profile. The two FPRs transduce very similar neutrophil responses but possess somewhat different regulatory profiles. The FPRs have served as excellent model receptors in studies attempting to understand not only GPCR related regulation in general, but also receptor signaling in relation to innate immune reactivity and inflammation. Recent research has identified not only a large number of conventional ligands (agonist/antagonists) that regulate FPR activities by binding to surface exposed parts of the receptors, but also a number of membrane penetrating molecules that allosterically modulate receptor function after passing the membrane and interacting with the receptor on the cytosolic side. After activation, FPR signaling is rapidly terminated and the receptors become desensitized, a dormant state that can be achieved by multiple mechanisms. A coupling of the activated receptors to the actin cytoskeleton in a process that physically separates the receptors from the signaling G-protein is one such mechanism. Traditionally, the desensitized state has been viewed as a point of no return, but recent findings challenge this view and demonstrate that desensitized FPRs may in fact be reactivated to resume active signaling. The FPRs have also the capacity to communicate with other receptors in a hierarchical manner and this receptor cross-talk can both dampen and amplify neutrophil responses. In this review, we summarize some recent advances of our understanding how the FPRs can be turned on and off and discuss some future challenges, including mechanisms of allosteric modulation, receptor cross-talk, and FPR reactivation

Turning chemoattractant receptors on and off with conventional ligands and allosteric modulators: recent advances in formyl peptide receptor signaling and regulation: DOI: 10.14800/ics.73

Recruitment and activation of neutrophils at sites of infection/inflammation relies largely on the surface expression of G-protein coupled receptors (GPCRs) that recognize chemoattractants. One of these receptors, FPR1, for which formylated peptides generated by bacteria and mitochondria are high affinity agonists, was among the first human neutrophil GPCR to being cloned. This receptor shares large sequence homologies with FPR2, another member of the FPR-family expressed in human neutrophils and having a distinct ligand binding profile. The two FPRs transduce very similar neutrophil responses but possess somewhat different regulatory profiles. The FPRs have served as excellent model receptors in studies attempting to understand not only GPCR related regulation in general, but also receptor signaling in relation to innate immune reactivity and inflammation. Recent research has identified not only a large number of conventional ligands (agonist/antagonists) that regulate FPR activities by binding to surface exposed parts of the receptors, but also a number of membrane penetrating molecules that allosterically modulate receptor function after passing the membrane and interacting with the receptor on the cytosolic side. After activation, FPR signaling is rapidly terminated and the receptors become desensitized, a dormant state that can be achieved by multiple mechanisms. A coupling of the activated receptors to the actin cytoskeleton in a process that physically separates the receptors from the signaling G-protein is one such mechanism. Traditionally, the desensitized state has been viewed as a point of no return, but recent findings challenge this view and demonstrate that desensitized FPRs may in fact be reactivated to resume active signaling. The FPRs have also the capacity to communicate with other receptors in a hierarchical manner and this receptor cross-talk can both dampen and amplify neutrophil responses. In this review, we summarize some recent advances of our understanding how the FPRs can be turned on and off and discuss some future challenges, including mechanisms of allosteric modulation, receptor cross-talk, and FPR reactivation

Systemic galectin-3 in smokers with chronic obstructive pulmonary disease and chronic bronchitis: The impact of exacerbations

Purpose: The carbohydrate-binding protein Galectin-3 is increased in several inflammatory diseases and has recently been forwarded as a systemic biomarker in chronic obstructive pulmonary disease (COPD). In this longitudinal study, we characterized the level of systemic Galectin-3 using blood from smokers with a history of COPD and chronic bronchitis (COPD-CB), during stable clinical conditions and exacerbations. Patients and Methods: The study population comprised 56 long-term smokers with COPD-CB, 10 long-term smokers without lung disease (LTS) and 10 clinically healthy never-smokers (HNS). Blood samples were analyzed for levels of Galectin-3, leukocyte populations and C-reactive protein (CRP). In addition, sputum samples from the COPD-CB group were analyzed for bacterial growth. Results: When comparing stable clinical conditions and exacerbations in the COPD-CB group, we found that the level of Galectin-3, just like that of CRP, leukocytes and neutrophils, respectively, was increased during exacerbations. However, this exacerbation-associated increase of Galectin-3 was modest. During stable clinical conditions of COPD-CB, the level of Galectin-3 was not elevated in comparison with HNS or LTS. Nor did this level of Galectin-3 distinguish patients that remained in a clinically stable condition throughout the study to those that developed an exacerbation. In addition, neither during stable clinical conditions nor during exacerbations, did the presence of bacterial growth in sputum alter Galectin-3 levels. In contrast to Galectin-3, the level of CRP, leukocytes and neutrophils, respectively, were increased during clinical stable conditions in the COPD-CB group compared with the other groups and were further enhanced during exacerbations. Conclusion: Systemic Galectin-3 is increased in a reproducible but modest manner during exacerbations in smokers with COPD-CB. During stable clinical conditions, the level of systemic Galectin-3 does not distinguish patients that remain clinically stable from those that develop exacerbations. This makes it less likely that systemic Galectin-3 may become a clinically useful biomarker in the current setting

Self-injury in youths who lost a parent to cancer: nationwide study of the impact of family-related and health-care-related factors.

BACKGROUND: Self-injury, a manifestation of severe psychological distress, is increased in cancer-bereaved youths. Little is known about the potential influence on the risk for self-injury of factors that could be clinically relevant to and modifiable by the health-care professionals involved in the care of the dying parent. METHODS: In a nationwide population-based anonymous study, 622 (73.1%) youths (aged 18-26) who, 6 to 9 years earlier at ages 13 to 16, had lost a parent to cancer answered study-specific questions about self-injury and factors related to the family and parental health care. RESULTS: Univariable analyses showed that the risk for self-injury was increased among cancer-bereaved youths who reported poor family cohesion the years before (relative risk [RR], 3.4, 95% confidence interval [CI], 2.5-4.6) and after the loss (RR, 3.3, 95% CI, 2.4-4.4), distrust in the health care provided to the dying parent (RR, 1.7, 95% CI, 1.2-2.4), perceiving poor health-care efforts to cure the parent (RR 1.5, 95% CI, 1.1-2.1) and poor efforts to prevent suffering (RR, 1.6, 95% CI, 1.1-2.4), that at least one of their parents had been depressed or had troubles in life (RR, 1.5, CI, 1.1-2.1) and believing 3 days before the loss that the treatment would probably cure the parent (RR, 1.6, CI, 1.1-2.3). In the total multivariable models, only poor family cohesion before and after the loss remained statistically significantly associated with self-injury. CONCLUSION: Poor family cohesion before and after the loss of a parent to cancer is associated with an increased risk of self-injury in teenage children

Teenagers want to be told when a parent's death is near: A nationwide study of cancer-bereaved youths' opinions and experiences.

BACKGROUND: We aimed to investigate cancer-bereaved youths' opinions and experiences of being told about a parent's imminent death from cancer and of barriers to this communication. MATERIAL AND METHODS: This nationwide population-based survey included 622/851 (73%) youths (aged 18-26) who at age 13-16, 6-9 years earlier had lost a parent to cancer. RESULTS: In total 595 of 610 (98%) of the participants stated that teenage children should be informed when the parent's death was imminent (i.e. a matter of hours or days, not weeks). 59% stated that they themselves had been told this, 37% by the parents, 7% by parents and healthcare professionals together and 8% by professionals only. Frequent reasons for why the teenager and parents did not talk about imminent death before loss were that one (n=106) or both (n=25) of the parents together with the teenage child had pretended that the illness was not that serious, or that none of the parents had been aware that death was imminent (n=80). Up to a couple of hours before the loss, 43% of participants had not realized that death was imminent. CONCLUSION: In this population-based study virtually all youth who at ages 13-16 had lost a parent to cancer afterwards stated that teenagers should be told when loss is near, i.e. a matter of hours or days, not weeks. Many stated that they had not been given this information and few were informed by professionals, with implications for future improvements in end-of-life care of patients with teenage children

Porphyromonas gingivalis Produce Neutrophil Specific Chemoattractants Including Short Chain Fatty Acids

Neutrophil migration from blood to tissue-residing microbes is governed by a series of chemoattractant gradients of both endogenous and microbial origin. Periodontal disease is characterized by neutrophil accumulation in the gingival pocket, recruited by the subgingival biofilm consisting mainly of gram-negative, anaerobic and proteolytic species such as Porphyromonas gingivalis. The fact that neutrophils are the dominating cell type in the gingival pocket suggests that neutrophil-specific chemoattractants are released by subgingival bacteria, but characterization of chemoattractants released by subgingival biofilm species remains incomplete. In the present study we characterized small (< 3 kDa) soluble chemoattractants released by growing P. gingivalis, and show that these are selective for neutrophils. Most neutrophil chemoattractant receptors are expressed also by mononuclear phagocytes, the free fatty acid receptor 2 (FFAR2) being an exception. In agreement with the selective neutrophil recruitment, the chemotactic activity found in P. gingivalis supernatants was mediated in part by a mixture of short chain fatty acids (SCFAs) that are recognized by FFAR2, and other leukocytes (including monocytes) did not respond to SCFA stimulation. Although SCFAs, produced by bacterial fermentation of dietary fiber in the gut, has previously been shown to utilize FFAR2, our data demonstrate that the pronounced proteolytic metabolism employed by P. gingivalis (and likely also other subgingival biofilm bacteria associated with periodontal diseases) may result in the generation of SCFAs that attract neutrophils to the gingival pocket. This finding highlights the interaction between SCFAs and FFAR2 in the context of P. gingivalis colonization during periodontal disease, but may also have implications for other inflammatory pathologies involving proteolytic bacteria

Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called "targeting by timing." Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, andN- andO-glycans present in each compartment were analyzed by LC-MS. We found abundant paucimannosidicN-glycans and lack ofO-glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complexN-andO-glycans with remarkably elongatedN-acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of "glycosylation by timing" during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function

Substantial Decrease in Comorbidity 5 Years After Gastric Bypass : A Population-based Study From the Scandinavian Obesity Surgery Registry

OBJECTIVE:: To evaluate effect on comorbid disease and weight loss 5 years after Roux-en-Y gastric bypass (RYGB) surgery for morbid obesity in a large nationwide cohort. BACKGROUND:: The number patients having surgical procedures to treat obesity and obesity-related disease are increasing. Yet, population-based, long-term outcome studies are few. METHODS:: Data on 26,119 individuals [75.8% women, 41.0 years, and body mass index (BMI) 42.8?kg/m] undergoing primary RYGB between May 1, 2007 and June 30, 2012, were collected from 2 Swedish quality registries: Scandinavian Obesity Surgery Registry and the Prescribed Drug Registry. Weight, remission of type 2 diabetes mellitus, hypertension, dyslipidemia, depression, and sleep apnea, and changes in corresponding laboratory data were studied. Five-year follow-up was 100% (9774 eligible individuals) for comorbid diseases. RESULTS:: BMI decreased from 42.8?±?5.5 to 31.2?±?5.5?kg/m at 5 years, corresponding to 27.7% reduction in total body weight. Prevalence of type 2 diabetes mellitus (15.5%–5.9%), hypertension (29.7%–19.5%), dyslipidemia (14.0%–6.8%), and sleep apnea (9.6%–2.6%) was reduced. Greater weight loss was a positive prognostic factor, whereas increasing age or BMI at baseline was a negative prognostic factor for remission. The use of antidepressants increased (24.1%–27.5%). Laboratory status was improved, for example, fasting glucose and glycated hemoglobin decreased from 6.1 to 5.4?mmol/mol and 41.8% to 37.7%, respectively. CONCLUSIONS:: In this nationwide study, gastric bypass resulted in large improvements in obesity-related comorbid disease and sustained weight loss over a 5-year period. The increased use of antidepressants warrants further investigation

Mice Chronically Fed High-Fat Diet Have Increased Mortality and Disturbed Immune Response in Sepsis

BACKGROUND: Sepsis is a potentially deadly disease that often is caused by gram-positive bacteria, in particular Staphylococcus aureus (S. aureus). As there are few effective therapies for sepsis, increased basic knowledge about factors predisposing is needed. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of this study was to study the effect of Western diet on mortality induced by intravenous S. aureus inoculation and the immune functions before and after bacterial inoculation. Here we show that C57Bl/6 mice on high-fat diet (HFD) for 8 weeks, like genetically obese Ob/Ob mice on low-fat diet (LFD), have increased mortality during S. aureus-induced sepsis compared with LFD-fed C57Bl/6 controls. Bacterial load in the kidneys 5-7 days after inoculation was increased 10-fold in HFD-fed compared with LFD-fed mice. At that time, HFD-fed mice had increased serum levels and fat mRNA expression of the immune suppressing cytokines interleukin-1 receptor antagonist (IL-1Ra) and IL-10 compared with LFD-fed mice. In addition, HFD-fed mice had increased serum levels of the pro-inflammatory IL-1beta. Also, HFD-fed mice with and without infection had increased levels of macrophages in fat. The proportion and function of phagocytosing granulocytes, and the production of reactive oxygen species (ROS) by peritoneal lavage cells were decreased in HFD-fed compared with LFD-fed mice. CONCLUSIONS: Our findings imply that chronic HFD disturb several innate immune functions in mice, and impairs the ability to clear S. aureus and survive sepsis

Glycoproteome remodeling and organelle-specific N-glycosylation accompany neutrophil granulopoiesis

Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we comprehensively profile the neutrophil N-glycoproteome with spatiotemporal resolution by analyzing four key types of intracellular organelles isolated from blood-derived neutrophils and during their maturation from bone marrow–derived progenitors using a glycomics-guided glycoproteomics approach. Interestingly, the organelles of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, highly truncated N-glycans low in α2,6-sialylation and Lewis fucosylation decorating a diverse set of microbicidal proteins (e.g., myeloperoxidase, azurocidin, neutrophil elastase) in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodeling underpins the promyelocytic-to-metamyelocyte transition and that the glycophenotypic differences are driven primarily by dynamic changes in protein expression and less by changes within the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with relatively high levels of glycosylation of the microbicidal proteins in the azurophilic granules. Our study provides spatiotemporal insights into the complex neutrophil N-glycoproteome featuring intriguing organelle-specific N-glycosylation patterns formed by dynamic glycoproteome remodeling during the early maturation stages of the myeloid progenitors