Regulation of the Formyl Peptide Receptor 1 (FPR1) Gene in Primary Human Macrophages

The formyl peptide receptor 1 (FPR1) is mainly expressed by mammalian phagocytic leukocytes and plays a role in chemotaxis, killing of microorganisms through phagocytosis, and the generation of reactive oxygen species. A large number of ligands have been identified triggering FPR1 including formylated and non-formylated peptides of microbial and endogenous origin. While the expression of FPR1 in neutrophils has been investigated intensively, knowledge on the regulation of FPR1 expression in polarized macrophages is lacking. In this study we show that primary human neutrophils, monocytes and resting macrophages do express the receptor on their cell surface. Polarization of macrophages with IFNγ, LPS and with the TLR8 ligand 3M-002 further increases FPR1 mRNA levels but does not consistently increase protein expression or chemotaxis towards the FPR1 ligand fMLF. In contrast, polarization of primary human macrophages with IL-4 and IL-13 leading to the alternative activated macrophages, reduces FPR1 cell surface expression and abolishes chemotaxis towards fMLF. These results show that M2 macrophages will not react to triggering of FPR1, limiting the role for FPR1 to chemotaxis and superoxide production of resting and pro-inflammatory M1 macrophages.     

Enterococcus faecium stimulates human neutrophils via the formyl-peptide receptor 2

The human formyl-peptide receptor 2 (FPR2/ALX) senses phenol-soluble modulin (PSM) peptide toxins produced by pathogenic staphylococcal species and plays a crucial role in directing neutrophil influx during staphylococcal infection. However, it has remained unclear if FPR2 responds also to molecules from other bacterial pathogens. Here we analyzed a variety of gram-positive and gram-negative pathogens and found that apart from staphylococci only certain enterococcal strains have the capacity to stimulate FPR2/ALX. Most of the analyzed Enterococcus faecium but only sporadic Enterococcus faecalis strains released FPR2/ALX-stimulating molecules leading to neutrophil calcium ion fluxes, chemotaxis, and complement receptor upregulation. Among ten test strains vancomycin-resistant E. faecium had a significantly higher capacity to stimulate FPR2/ALX than vancomycin-susceptible strains, suggesting an association of strong FPR2/ALX activation with health-care associated strains. The enterococcal FPR2/ALX agonists were found to be peptides or proteins, which appear, however, to be unrelated to staphylococcal PSMs in sequence and physicochemical properties. Enterococci are among the most frequent invasive bacterial pathogens but the basis of enterococcal virulence and immune activation has remained incompletely understood. Our study indicates that previously unrecognized proteinaceous agonists contribute to Enterococcus-host interaction and underscores the importance of FPR2/ALX in host defense against major endogenous bacterial pathogens.     

N-formyl peptide receptor 3 (FPR3) departs from the homologous FPR2/ALX receptor with regard to the major processes governing chemoattractant receptor regulation, expression at the cell surface, and phosphorylation

Among human N-formyl peptide chemoattractant receptors, FPR2/ALX and FPR3 share the highest degree of amino acid identity (83%), and trigger similar cell responses upon ligand binding. Although FPR2/ALX is a promiscuous receptor, FPR3 has only one specific high affinity ligand, F2L, and a more restricted tissue/cell distribution. In this study, we showed that FPR2/ALX behaved as the prototypical receptor FPR1. The agonist-dependent phosphorylation used a hierarchical mechanism with a prominent role of Ser(329), Thr(332), and Thr(335). Phosphorylation of FPR2/ALX was essential for its desensitization but the lack of phosphorylation did not result in enhanced or sustained responses. In contrast, resting FPR3 displayed a marked level of phosphorylation, which was only slightly increased upon agonist stimulation. Another noticeable difference between the two receptors was their subcellular distribution in unstimulated cells. Although FPR2/ALX was evenly distributed at the plasma membrane FPR3 was localized in small intracellular vesicles. By swapping domains between FPR2/ALX and FPR3, we uncovered the determinants involved in the basal phosphorylation of FPR3. Experiments aimed at monitoring receptor-bound antibody uptake showed that the intracellular distribution of FPR3 resulted from a constitutive internalization that was independent of C terminus phosphorylation. Unexpectedly, exchanging residues 1 to 53, which encompass the N-terminal extracellular region and the first transmembrane domain, between FPR2/ALX and FPR3 switched localization of the receptors from the plasma membrane to intracellular vesicles and vice versa. A clathrin-independent, possibly caveolae-dependent, mechanism was involved in FPR3 constitutive internalization. The peculiar behavior of FPR3 most probably serves distinct physiological functions that remain largely unknown.     

Promoting effect of IFN-γ on the expression of LPS-induced IL-12 p40 and p35 mRNA in murine suppressor macrophages

We detected the expression of IL-12 p40/p35 mRNA by semi-quantitative RT-PCR and silver staining, and studied the molecular interaction between the IL-12 expression and the NF-kB activation induced by LPS and IFN-γ/LPS in murine peritoneal suppressor macrophages (MPSMs). It was found that IFN-γ strongly enhanced the LPS-induced IL-12 p40 and p35 mRNA expression. Both p40 and p35 mRNA levels were approximately equal. IFN-γ also greatly promoted the LPS-induced secretion of IL-12 p70 in MPSMs. The Proteasome Inhibitor I (PSI) could block the expres-sion of IL-12 p40 and p35 mRNA, and the degradation of IkBα induced by LPS or LPS/IFN-γ. EM-SA showed that LPS could augment the NF-kB binding activity to p40 promoter DNA. However, IFN-γ could neither enhance the LPS-induced NF-kB activity nor promote the degradation of IkBα. Taken together, the data suggest: (i) IFN-γ/LPS could strongly induce the expression of IL-12 p40 and p35 mRNA; both the expression levels were equal; this phenomenon coincided with the high-level secretion of IL-12 p70 induced by IFN-γ/LPS; (ii) NF-kB signal pathway is essential for IFN-γ/LPS to induce IL-12 mRNA expression; (iii) by blocking the degradation of IkB, the PSI sup-presses the IL-12 p40/p35 mRNA expression induced by LPS and IFN-γ/LPS; (iv) NF-kB signal may not be involved in the mechanism by which IFN-γ enhanced the expression of the LPS-induced IL-12 p40/p35 mRNA.     

Resolvin D1 prevents TNF-α-mediated disruption of salivary epithelial formation

Sj?gren's syndrome is a chronic autoimmune disorder characterized by inflammation of salivary glands resulting in impaired secretory function. Our present studies indicate that chronic exposure of salivary epithelium to TNF-α and/or IFN-γ alters tight junction integrity, leading to secretory dysfunction. Resolvins of the D-series (RvDs) are endogenous lipid mediators derived from DHA that regulate excessive inflammatory responses leading to resolution and tissue homeostasis. In this study, we addressed the hypothesis that activation of the RvD1 receptor ALX/FPR2 in salivary epithelium prevents and/or resolves the TNF-α-mediated disruption of acinar organization and enhances monolayer formation. Our results indicate that 1) the RvD1 receptor ALX/FPR2 is present in fresh, isolated cells from mouse salivary glands and in cell lines of salivary origin; and 2) the agonist RvD1 (100 ng/ml) abolished tight junction and cytoskeletal disruption caused by TNF-α and enhanced cell migration and polarity in salivary epithelium. These effects were blocked by the ALX/FPR2 antagonist butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe. The ALX/FPR2 receptor signals via modulation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways since, in our study, blocking PI3K activation with LY294002, a potent and selective PI3K inhibitor, prevented RvD1-induced cell migration. Furthermore, Akt gene silencing with the corresponding siRNA almost completely blocked the ability of Par-C10 cells to migrate. Our findings suggest that RvD1 receptor activation promotes resolution of inflammation and tissue repair in salivary epithelium, which may have relevance in the restoration of salivary gland dysfunction associated with Sj?gren's syndrome.     

Posttranscriptional regulation of IL-23 expression by IFN-gamma through tristetraprolin

IL-23 plays an essential role in maintenance of IL-17-producing Th17 cells that are involved in the pathogenesis of several autoimmune diseases. Regulation of Th17 cells is tightly controlled by multiple factors such as IL-27 and IFN-γ. However, the detailed mechanisms responsible for IFN-γ-mediated Th17 cell inhibition are still largely unknown. In this study, we demonstrate that IFN-γ differentially regulates IL-12 and IL-23 production in both dendritic cells and macrophages. IFN-γ suppresses IL-23 expression by selectively targeting p19 mRNA stability through its 3'-untranslated region (3'UTR). Furthermore, IFN-γ enhances LPS-induced tristetraprolin (TTP) mRNA expression and protein production. Overexpression of TTP suppresses IL-23 p19 mRNA expression and p19 3'UTR-dependent luciferase activity. Additionally, deletion of TTP completely abolishes IFN-γ-mediated p19 mRNA degradation. We further demonstrate that IFN-γ suppresses LPS-induced p38 phosphorylation, and blockade of p38 MAPK signaling pathway with SB203580 inhibits IFN-γ- and LPS-induced p19 mRNA expression, whereas overexpression of p38 increases p19 mRNA expression via reducing TTP binding to the p19 3'UTR. Finally, inhibition of p38 phosphorylation by IFN-γ leads to TTP dephosphorylation that could result in stronger binding of the TTP to the adenosine/uridine-rich elements in the p19 3'UTR and p19 mRNA degradation. In summary, our results reveal a direct link among TTP, IFN-γ, and IL-23, indicating that IFN-γ-mediated Th17 cell suppression might act through TTP by increasing p19 mRNA degradation and therefore IL-23 inhibition.     

Annexin A1 interaction with the FPR2/ALX receptor: identification of distinct domains and downstream associated signaling

Understanding how proresolving agonists selectively activate FPR2/ALX is a crucial step in the clarification of proresolution molecular networks that can be harnessed for the design of novel therapeutics for inflammatory disease. FPR2/ALX, a G protein-coupled receptor belonging to the formyl peptide receptor (FPR) family, conveys the biological functions of a variety of ligands, including the proresolution mediators annexin A1 (AnxA1) and lipoxin A(4), as well as the activating and proinflammatory protein serum amyloid A. FPR2/ALX is the focus of intense screening for novel anti-inflammatory therapeutics, and the small molecule compound 43 was identified as a receptor ligand. Here, we used chimeric FPR1 and FPR2/ALX clones (stably transfected in HEK293 cells) to identify the N-terminal region and extracellular loop II as the FPR2/ALX domain required for AnxA1-mediated signaling. Genomic responses were also assessed with domain-specific effects emerging, so the N-terminal region is required for AnxA1 induction of JAG1 and JAM3, whereas it is dispensable for modulation of SGPP2. By comparison, serum amyloid A non-genomic responses were reliant on extracellular loops I and II, whereas the small molecule compound 43 activated extracellular loop I with downstream signaling dependent on transmembrane region II. In desensitization experiments, the N-terminal region was dispensable for AnxA1-induced FPR2/ALX down-regulation in both the homologous and heterologous desensitization modes.     

Brucella abortus inhibits IFN-γ-induced FcγRI expression and FcγRI-restricted phagocytosis via toll-like receptor 2 on human monocytes/macrophages

The strategies that allow Brucella abortus to persist for years inside macrophages subverting host immune responses are not completely understood. Immunity against this bacterium relies on the capacity of IFN-γ to activate macrophages, endowing them with the ability to destroy intracellular bacteria. We report here that infection with B. abortus down-modulates the expression of the type I receptor for the Fc portion of IgG (FcγRI, CD64) and FcγRI-restricted phagocytosis regulated by IFN-γ in human monocytes/macrophages. Both phenomena were not dependent on bacterial viability, since they were also induced by heat-killed B. abortus (HKBA), suggesting that they were elicited by a structural bacterial component. Accordingly, a prototypical B. abortus lipoprotein (L-Omp19), but not its unlipidated form, inhibited both CD64 expression and FcγRI-restricted phagocytosis regulated by IFN-γ. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited CD64 expression, indicating that any Brucella lipoprotein could down-modulate CD64 expression and FcγRI-restricted phagocytosis. Pre-incubation of monocytes/macrophages with anti-TLR2 mAb blocked the inhibition of the CD64 expression mediated by HKBA and L-Omp19. These results, together with our previous observations establish that B. abortus utilizes its lipoproteins to inhibit the monocytes/macrophages activation mediated by IFN-γ and to subvert host immunonological responses.     

Resolvin D1 inhibits TGF-β1-induced epithelial mesenchymal transition of A549 lung cancer cells via lipoxin A4 receptor/formyl peptide receptor 2 and GPR32

Epithelial-mesenchymal-transition (EMT) is a key event for tumor cells to initiate metastasis which lead to switching of E-cadherin to N-cadherin. Resolvins are known to promote the resolution of inflammation and phagocytosis of macrophages. However, the role of resolvins in EMT of cancer is not known. Therefore, we examined the effects of resolvins on transforming growth factor, beta 1 (TGF-β1)-induced EMT. Expression of E-cadherin and N-cadherin in A549 lung cancer cells was evaluated by Western blot and confocal microscopy. Involvement of lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2) was examined by gene silencing. TGF-β1 induced expression of N-cadherin in A549 lung cancer cells, and resolvin D1 and D2 inhibited the expression of N-cadherin at low concentrations (1–100 nM). Resolvin D1 and D2 also suppressed the expression of zinc finger E-box binding homeobox 1 (ZEB1). The effects of resolvin D1 and D2 were confirmed in other lung cancer cell lines such as H838, H1299, and H1703. Resolvin D1 and D2 did not affect the proliferation of A549 lung cancer cells. Resolvin D1 and D2 also suppressed the TGF-β1-induced morphological change. Resolvin D1 and D2 also inhibited the TGF-β1-induced migration and invasion of A549 cells. Resolvin D1 is known to act via ALX/FPR2 and GPR32. Thus, we examined the involvement of ALX/FPR2 and GPR32 in the suppressive effects of resolvin D1 on TGF-β1-induced EMT of A549 cells. Gene silencing of ALX/FPR2 and GPR32 blocked the action of resolvin D1. Overexpression of ALX/FPR2 or GPR32 increased the effects of resolvin D1. These results suggest that resolvin D1 inhibited TGF-β1-induced EMT via ALX/FPR2 and GPR32 by reducing the expression of ZEB1.     

PPARγ stabilizes HO-1 mRNA in monocytes/macrophages which affects IFN-β expression

NADPH oxidase activation in either RAW264.7 cells or peritoneal macrophages (PM) derived from PPARγ wild-type mice increased reactive oxygen species (ROS) formation, caused PPARγ activation, heme oxygenase-1 (HO-1) induction, and concomitant IFN-β expression. In macrophages transduced with a dominant negative (d/n) mutant of PPARγ (RAW264.7 AF2) as well as PPARγ negative PM derived from Mac-PPARγ-KO mice, NADPH oxidase-dependent IFN-β expression was attenuated. As the underlying mechanism, we noted decreased HO-1 mRNA stability in RAW264.7 AF2 cells as well as PPARγ negative PM, compared to either parent RAW264.7 cells or wild-type PM. Assuming mRNA stabilization of HO-1 by PPARγ we transfected macrophages with a HO-1 3′-UTR reporter construct. The PPARγ agonist rosiglitazone significantly up-regulated luciferase expression in RAW264.7 cells, while it remained unaltered in RAW264.7 AF2 macrophages. Deletion of each of two AU-rich elements in the 3′-UTR HO-1 decreased luciferase activity in RAW264.7 cells. Using LPS as a NADPH oxidase activator, PM from Mac-PPARγ-KO mice showed a decreased HO-1 mRNA half-life in vitro and in vivo compared to PPARγ wild-type mice. These data identified a so far unappreciated role of PPARγ in stabilizing HO-1 mRNA, thus, contributing to the expression of the HO-1 target gene IFN-β.     

Macrophage sub-populations and the lipoxin A4 receptor implicate active inflammation during equine tendon repair

Macrophages (Mφ) orchestrate inflammatory and reparatory processes in injured connective tissues but their role during different phases of tendon healing is not known. We investigated the contribution of different Mφ subsets in an equine model of naturally occurring tendon injury. Post mortem tissues were harvested from normal (uninjured), sub-acute (3-6 weeks post injury) and chronically injured (>3 months post injury) superficial digital flexor tendons. To determine if inflammation was present in injured tendons, Mφ sub-populations were quantified based on surface antigen expression of CD172a (pan Mφ), CD14(high)CD206(low) (pro-inflammatory M1Mφ), and CD206(high) (anti-inflammatory M2Mφ) to assess potential polarised phenotypes. In addition, the Lipoxin A(4) receptor (FPR2/ALX) was used as marker for resolving inflammation. Normal tendons were negative for both Mφ and FPR2/ALX. In contrast, M1Mφ predominated in sub-acute injury, whereas a potential phenotype-switch to M2Mφ polarity was seen in chronic injury. Furthermore, FPR2/ALX expression by tenocytes was significantly upregulated in sub-acute but not chronic injury. Expression of the FPR2/ALX ligand Annexin A1 was also significantly increased in sub-acute and chronic injuries in contrast to low level expression in normal tendons. The combination of reduced FPR2/ALX expression and persistence of the M2Mφ phenotype in chronic injury suggests a potential mechanism for incomplete resolution of inflammation after tendon injury. To investigate the effect of pro-inflammatory mediators on lipoxin A(4) (LXA(4)) production and FPR2/ALX expression in vitro, normal tendon explants were stimulated with interleukin-1 beta and prostaglandin E(2). Stimulation with either mediator induced LXA(4) release and maximal upregulation of FPR2/ALX expression after 72 hours. Taken together, our data suggests that although tenocytes are capable of mounting a protective mechanism to counteract inflammatory stimuli, this appears to be of insufficient duration and magnitude in natural tendon injury, which may potentiate chronic inflammation and fibrotic repair, as indicated by the presence of M2Mφ.