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

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

Receptor Cross-talk and Neutrophil Function

The chemotactic recruitment of neutrophils, the most abundant white blood cell in human circulation, to sites of infection and inflammation, is dependent upon a gradient of chemoattractants released from cells at the inflamed area. The chemoattractants, including formyl peptides, are recognized by G-protein coupled chemoattractant receptors (GPCRs) present on the neutrophil surface. Activation of GPCRs in neutrophils mediates chemotaxis, but also granule mobilization and the generation of reactive oxygen species (ROS). ROS and granule constituents are not only essential for effective microbial killing, but may also account for unwanted tissue destruction. Stringent activation and termination of neutrophil GPCR signaling is therefore crucial for fine-tuning of inflammatory reactions. Two well-known control mechanisms are 1) receptor desensitization, a non-signaling state reached after termination of the agonist-induced GPCR signal, and 2) priming, a hyper-responsive state involving upregulation of surface receptors. The data presented in this thesis explore both of these control mechanisms, and in addition provide evidence for the existence of a novel receptor cross-talk mechanism whereby already desensitized receptors can be reactivated. We first show that in analogy to what is known for human neutrophils, TNF-α is able to prime mouse neutrophils for FPR stimulation. Next we show that FPR desensitization can be broken by treatment with the b-galactioside biding human lectin galectin-3. This process is dependent upon ROS-mediated inactivation of the FPR agonist, which in turn relies on the carbohydrate-binding domain of the lectin and on the presence of the neutrophil peroxidase MPO. Most importantly, this thesis also discovers a novel cross talk mechanism whereby desensitized FPRs can be reactivated and turned into active signaling. We show that stimulation of FPR desensitized neutrophils with 1) extracellular ATP (a damage-associated molecular pattern; DAMP) and 2) the platelet activating factor (PAF) transmit signals leading to reactivation of FPRs. This could be an important mechanism for amplification of cellular responsiveness during contact with multiple inflammatory mediators simultaneously. The signals leading to FPR reactivation were shown to be independent of intracellular calcium signaling, and an intact actin cytoskeleton, but required calyculinA-sensitive phosphatases. The data presented challenge the current view of actin-dependent FPR desensitization and the view of the desensitization process as a stable point-of-no-return

Data on the NADPH-oxidase activity induced by WKYMVm and galectin-3 in bone marrow derived and exudated neutrophils isolated from four different mouse strains

Neutrophils are the key players in inflammatory reactions and the release of superoxide through the NADPH-oxidase upon neutrophil activation contributes to bacterial clearance and surrounding tissue damage. Here we describe data on the mouse neutrophil NADPH-oxidase activation induced by the mouse formyl peptide receptor (Fpr) agonist WKYMVm and galectin-3. Neutrophils isolated from bone marrow, peritoneal exudated, and in vitro TNFα primed bone marrow neutrophils from four different laboratory strains (C57BL/6, DBA/1, BALB/c and NMRI) were used. Both Fpr agonist and galectin-3 activated neutrophils to release superoxide. No differences were observed in the amounts of superoxide released from neutrophils derived from four different strains

Vaccination with the Secreted Glycoprotein G of Herpes Simplex Virus 2 Induces Protective Immunity after Genital Infection

Herpes simplex virus 2 (HSV-2) infects the genital mucosa and establishes a life-long infection in sensory ganglia. After primary infection HSV-2 may reactivate causing recurrent genital ulcerations. HSV-2 infection is prevalent, and globally more than 400 million individuals are infected. As clinical trials have failed to show protection against HSV-2 infection, new vaccine candidates are warranted. The secreted glycoprotein G (sgG-2) of HSV-2 was evaluated as a prophylactic vaccine in mice using two different immunization and adjuvant protocols. The protocol with three intramuscular immunizations combining sgG-2 with cytosine-phosphate-guanine dinucleotide (CpG) motifs and alum induced almost complete protection from genital and systemic disease after intra-vaginal challenge with HSV-2. Robust immunoglobulin G (IgG) antibody titers were detected with no neutralization activity. Purified splenic CD4+ T cells proliferated and produced interferon-γ (IFN-γ) when re-stimulated with the antigen in vitro. sgG-2 + adjuvant intra-muscularly immunized mice showed a significant reduction of infectious HSV-2 and increased IFN-γ levels in vaginal washes. The HSV-2 DNA copy numbers were significantly reduced in dorsal root ganglia, spinal cord, and in serum at day six or day 21 post challenge. We show that a sgG-2 based vaccine is highly effective and can be considered as a novel candidate in the development of a prophylactic vaccine against HSV-2 infection

Receptor cross talk from the PAFR induces reactivation of FPR1_des.

<p>Human neutrophils (10⁵) were desensitized with the FPR1 agonist fMIFL (0.1 nM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060169#pone-0060169-g001" target="_blank">Figure 1</a>. (<b>A</b>) The FPR1_des neutrophils were activated with PAF (100 nM, added at time indicated by arrow; solid line). The involvment of FPR1 and PAFR in the PAF-induced response was examined by addition of cyclosporin H (1 µM, FPR1 antagonist, broken line) or WEB2086 (1 µM, PAFR antagonist, dotted line) at 3 min prior to PAF addition. For comparison, the oxidative response to PAF in naïve neutrophils is shown (inset). A representative experiment is shown, n>5. Abscissa, time of study (min); Ordinate, superoxide production (counts per minute×10⁶; Mcpm). (<b>B</b>) Inhibition of the PAF-induced response in FPR1_des cells by cyclosporin H (1 µM, FPR1 specific antagonist) or WEB2086 (1 µM, PAFR antagonist) shown as mean peak values ±SEM of the responses (Mcpm, n = 5 for WEB2086, n = 19 for control, cyclosporine H). The PAF induced response in naïve neutrophils is shown for comparison (n = 19). (<b>C</b>) Human neutrophils (10⁵) were activated/desensitized with different concentrations of the FPR1 agonist fMIFL (added at time indicated by arrow to the left). The neutrophils were then activated with PAF (100 nM final concentration, added at time indicated by arrow to the right). For comparison, a PAF-induced response in naïve neutrophils is shown (solid line). A representative experiment is shown, n>5. Abscissa, time of study (min); Ordinate, superoxide production (counts per minute×10⁶; Mcpm).</p

Reactivation of Desensitized Formyl Peptide Receptors by Platelet Activating Factor: A Novel Receptor Cross Talk Mechanism Regulating Neutrophil Superoxide Anion Production

<div><p>Neutrophils express different chemoattractant receptors of importance for guiding the cells from the blood stream to sites of inflammation. These receptors communicate with one another, a cross talk manifested as hierarchical, heterologous receptor desensitization. We describe a new receptor cross talk mechanism, by which desensitized formyl peptide receptors (FPR_des) can be reactivated. FPR desensitization is induced through binding of specific FPR agonists and is reached after a short period of active signaling. The mechanism that transfers the receptor to a non-signaling desensitized state is not known, and a signaling pathway has so far not been described, that transfers FPR_des back to an active signaling state. The reactivation signal was generated by PAF stimulation of its receptor (PAFR) and the cross talk was uni-directional. LatrunculinA, an inhibitor of actin polymerization, induced a similar reactivation of FPR_des as PAF while the phosphatase inhibitor CalyculinA inhibited reactivation, suggesting a role for the actin cytoskeleton in receptor desensitization and reactivation. The activated PAFR could, however, reactivate FPR_des also when the cytoskeleton was disrupted prior to activation. The receptor cross talk model presented prophesies that the contact on the inner leaflet of the plasma membrane that blocks signaling between the G-protein and the FPR is not a point of no return; the receptor cross-talk from the PAFRs to the FPR_des initiates an actin-independent signaling pathway that turns desensitized receptors back to a signaling state. This represents a novel mechanism for amplification of neutrophil production of reactive oxygen species.</p> </div

Phosphatase inhibition by CalyculinA has both inhibitory and priming effects on the neutrophil NADPH-oxidase response.

<p>(<b>A</b>) Human neutrophils were incubated without or with CalyculinA (CA; 60 nM) at 37°C for 10 min prior to stimulation with PAF (100 nM) or fMIFL (0.1 nM), and the release of superoxide anions was recorded. The graph shows ratios of superoxide production induced by PAF or fMLF between samples with and without calyculin A (fold increase, mean ±SEM; n = 5). (<b>B</b>) FPR1_des neutrophils (desensitized with 0.1 nM fMIFL) were incubated at 37°C for 10 min without (control and inset, solid line) or with CalyculinA (CA, 50 nM; inset, broken line). The cells were then stimulated with PAF (100 nM) or latrunculin A (100 ng/ml final concentration) and the release of superoxide anions was recorded. A representative experiment for PAF stimulation is shown in the inset. The stimulus-induced responses in the CalyculinA treated FPR1_des neutrophils are expressed as percent of non-treated controls and is given as means ±SEM (n = 8).</p

Characteristics of the receptor antagonists used.

<p>Characteristics of the receptor antagonists used.</p