Prostaglandin E2 inhibits specific lung fibroblast functions via selective actions of PKA and Epac-1

Via their capacities for proliferation and synthesis of matrix proteins such as collagen, fibroblasts are key effectors in the pathogenesis of fibrotic disorders such as idiopathic pulmonary fibrosis. Prostaglandin E(2) (PGE(2)) potently inhibits these functions in lung fibroblasts through receptor ligation and production of the second messenger cAMP, but the downstream pathways mediating such actions have not been fully characterized. We sought to investigate the roles of the cAMP effectors protein kinase A (PKA) and exchange protein activated by cAMP-1 (Epac-1) in modulating these two functions in primary human fetal lung IMR-90 fibroblasts. The specific roles of these two effector pathways were examined by treating cells with PKA-specific (6-bnz-cAMP) and Epac-specific (8-pCPT-2'-O-Me-cAMP) agonists, inhibiting PKA with the inhibitor KT 5720, overexpressing the PKA catalytic subunit, and silencing Epac-1 using short hairpin RNA. PGE(2) inhibition of collagen I expression was mediated exclusively by activation of PKA, while inhibition of fibroblast proliferation was mediated exclusively by activation of Epac-1. PGE(2) and Epac-1 inhibited cell proliferation through activation of the small GTPase Rap1, since decreasing Rap1 activity by transfection with Rap1GAP or the dominant-negative Rap1N17 prevented, and transfection with the constitutively active Rap1V12 mimicked, the anti-proliferative effects of PGE(2). On the other hand, PKA inhibition of collagen was dependent on inhibition of protein kinase C-delta. The selective use of PKA and Epac-1 pathways to inhibit distinct aspects of fibroblast activation illustrate the pleiotropic ability of PGE(2) to inhibit diverse fibroblast functions.     

beta-Agonist enhances type 2 T-cell survival and accumulation

BACKGROUND: Neurohumoral modulation of immune system function is poorly understood. beta-Adrenergic receptor ligands (beta-agonists) subserve numerous physiologic processes but also function as pathogenic or therapeutic agents in numerous diseases with inflammatory components. OBJECTIVES: We sought to establish the effects of beta-agonists and prostaglandin E(2) (PGE(2)) on antigen-dependent and antigen-independent accumulation of IL-13(+) (type 2) and IFN-gamma(+) (type 1) T cells. We also sought to clarify the mechanisms mediating the effects of these G protein-coupled receptor agonists. METHODS: Effects of beta-agonists or PGE(2) on T-cell subtype accumulation were assessed in peripheral blood lymphocytes cultured with alphaCD3/CD28 or IL-2 by using flow cytometry. The role of cyclic AMP-dependent protein kinase (PKA) in mediating agonist effects was assessed by means of characterization of (1) phosphorylation of an intracellular PKA substrate and (2) T cells from patients with lupus possessing a natural defect in PKA activation. RESULTS: beta-Agonists, in contrast to PGE(2), increased IL-2-induced accumulation of human type 2 T cells, an effect attributable to differential activation of PKA affecting regulation of cell proliferation and apoptosis. In T cells from patients with lupus exhibiting defective PKA activation, both beta-agonists and PGE(2) promoted an increase in type 2 T-cell accumulation. CONCLUSION: G(s)-coupled receptors have the capacity to elicit prosurvival signaling in type 2 T cells, which, in most instances, is obscured by concomitant and antimitogenic PKA activation. CLINICAL IMPLICATIONS: beta-Agonists and other G(s)-coupled receptor agonists have the potential to regulate T-cell development to affect disease pathogenesis or the efficacy of therapies, and variability of effect relates to the ability to stimulate PKA activity.     

Paeoniflorin suppresses inflammatory mediator production and regulates G protein-coupled signaling in fibroblast – like synoviocytes of collagen induced arthritic rats

OBJECTIVE: To investigate the effects of Paeoniflorin (Pae) on inflammatory mediators and G protein - coupled signaling in fibroblast - like synoviocytes (FLS) from collagen induced arthritic (CIA) rats. METHODS: SD rats were injected with type II collagen. Pae (25, 50, 100 mg. kg(-1)) was administered to CIA rats. The inflammation of CIA rats was evaluated by paw swelling, arthritis index and histopathology of joints. FLS were isolated and cultured. Interleukin (IL)-1 activity was measured by the (3)H-TdR - intake method Tumor necrosis factor alpha (TNF-alpha), prostaglandin E(2) (PGE(2)) and cAMP were measured by radioimmunoassay. Protein kinase A (PKA) was assessed by luminescent kinase assay. Gi was detected by Western blot. RESULTS: Inflammation in CIA rats was accompanied by hyperplastic synovium, pannus and cartilage erosion in joints. IL-1 activity and Gi expression increased, PGE(2) and TNF-alpha production were enhanced, but cAMP level and PKA activity decreased. Pae significantly suppressed the inflammatory response and inflammatory mediators (IL-1, TNF-alpha and PGE(2)) in vivo. Pae inhibited Gi expression and restored cAMP level and PKA activity in FLS of CIA rats in vivo and vitro. CONCLUSION: Inflammatory mediators and G protein - coupled signaling were associated with the pathogenesis of synovitis in CIA rats. Pae, as a new monomer, had anti-inflammatory effects on the animal model of CIA in rats, but also had regulatory effects on FLS from CIA rats in vitro. These results highlight Pae as a good candidate for therapeutic intervention in RA.     

The Tick Salivary Protein Sialostatin L2 Inhibits Caspase-1-Mediated Inflammation during Anaplasma phagocytophilum Infection

Saliva from arthropod vectors facilitates blood feeding by altering host inflammation. Whether arthropod saliva counters inflammasome signaling, a protein scaffold that regulates the activity of caspase-1 and cleavage of interleukin-1β (IL-1β) and IL-18 into mature molecules, remains elusive. In this study, we provide evidence that a tick salivary protein, sialostatin L2, inhibits inflammasome formation during pathogen infection. We show that sialostatin L2 targets caspase-1 activity during host stimulation with the rickettsial agent Anaplasma phagocytophilum. A. phagocytophilum causes macrophage activation and hemophagocytic syndrome features. The effect of sialostatin L2 in macrophages was not due to direct caspase-1 enzymatic inhibition, and it did not rely on nuclear factor κB or cathepsin L signaling. Reactive oxygen species from NADPH oxidase and the Loop2 domain of sialostatin L2 were important for the regulatory process. Altogether, our data expand the knowledge of immunoregulatory pathways of tick salivary proteins and unveil an important finding in inflammasome biology.     

ATP binding by NLRP7 is required for inflammasome activation in response to bacterial lipopeptides

Nucleotide-binding oligimerization domain (NOD)-like receptors (NLRs) are pattern recognition receptors (PRRs) involved in innate immune responses. NLRs encode a central nucleotide-binding domain (NBD) consisting of the NAIP, CIITA, HET-E and TP1 (NACHT) domain and the NACHT associated domain (NAD), which facilitates receptor oligomerization and downstream inflammasome signaling. The NBD contains highly conserved regions, known as Walker motifs, that are required for nucleotide binding and hydrolysis. The NLR containing a PYRIN domain (PYD) 7 (NLRP7) has been recently shown to assemble an ASC and caspase-1-containing high molecular weight inflammasome complex in response to microbial acylated lipopeptides and Staphylococcus aureus infection. However, the molecular mechanism responsible for NLRP7 inflammasome activation is still elusive. Here we demonstrate that the NBD of NLRP7 is an ATP binding domain and has ATPase activity. We further show that an intact nucleotide-binding Walker A motif is required for NBD-mediated nucleotide binding and hydrolysis, oligomerization, and NLRP7 inflammasome formation and activity. Accordingly, THP-1 cells expressing a mutated Walker A motif display defective NLRP7 inflammasome activation, interleukin (IL)-1β release and pyroptosis in response to acylated lipopeptides and S. aureus infection. Taken together, our results provide novel insights into the mechanism of NLRP7 inflammasome assembly.     

Lipopolysaccharide (LPS) Induces the Apoptosis and Inhibits Osteoblast Differentiation Through JNK Pathway in MC3T3-E1 Cells

Bone degradation is a serious complication of chronic inflammatory diseases such as septic arthritis, osteomyelitis, and infected orthopedic implant failure. Up to date, effective therapeutic treatments for bacteria-caused bone destruction are limited. In our previous study, we found that LPS promoted osteoclast differentiation and activity through activation of mitogen-activated protein kinases (MAPKs) pathway such as c-Jun N-terminal kinases (JNK) and extracellular signal regulated kinase (ERK1/2). The current study was to evaluate the mechanism of LPS on the apoptosis and osteoblast differentiation in MC3T3-E1 cells. MC3T3-E1 osteoblasts were non-treated, treated with LPS. After treatment, the cell viability, the activity of alkaline phosphatase (ALP) and caspase-3 were measured. The expressions of osteoblast-specific genes and Bax, Bcl-2, and caspase-3 were determined by real-time quantitative polymerase chain reaction (qPCR). Protein levels of Bax, Bcl-2, caspase-3, and phosphorylation of MAPKs were measured using Western blotting assays. The MAPK signaling pathway was blocked by pretreatment with JNK inhibitor SP600125. LPS treatment induced a significant decrease in cell metabolism, viability, and ALP activity in MC3T3-E1 cells. LPS also significantly decreased mRNA expressions of osteoblast-related genes in MC3T3-E1 cells. On the other hand, LPS significantly upregulated mRNA expressions and protein levels of Bax and caspase-3 as well as activation of caspase-3, whereas decreased Bcl-2 expression in MC3T3-E1 cells. Furthermore, LPS significantly promoted MAPK pathway including the phosphorylation of JNK and the phosphorylation of ERK1/2; moreover, pretreatment with JNK inhibitor not only attenuated both of phosphorylation-JNK and ERK1/2 enhanced by LPS in MC3T3-E1 cells, but also reversed the downregulated expressions of osteoblast-specific genes including ALP and BSP induced by LPS. In conclusion, LPS could induce osteoblast apoptosis and inhibit osteoblast differentiation via activation of JNK pathway.     

Pannexin-1-mediated recognition of bacterial molecules activates the cryopyrin inflammasome independent of Toll-like receptor signaling

Cryopyrin is essential for caspase-1 activation triggered by Toll-like receptor (TLR) ligands in the presence of adenosine triphosphate (ATP). However, the events linking bacterial products and ATP to cryopyrin remain unclear. Here we demonstrate that cryopyrin-mediated caspase-1 activation proceeds independently of TLR signaling, thus dissociating caspase-1 activation and IL-1beta secretion. Instead, caspase-1 activation required pannexin-1, a hemichannel protein that interacts with the P2X(7) receptor. Direct cytosolic delivery of multiple bacterial products including lipopolysaccharide, but not flagellin, induced caspase-1 activation via cryopyrin in the absence of pannexin-1 activity or ATP stimulation. However, unlike Ipaf-dependent caspase-1 activation, stimulation of the pannexin-1-cryopyrin pathway by several intracellular bacteria was independent of a functional bacterial type III secretion system. These results provide evidence for cytosolic delivery and sensing of bacterial molecules as a unifying model for caspase-1 activation and position pannexin-1 as a mechanistic link between bacterial stimuli and the cryopyrin inflammasome.     

Molecular mechanisms of inflammasome activation during microbial infections

The innate immune system plays a crucial role in the rapid recognition and elimination of invading microbes. Detection of microbes relies on germ-line encoded pattern recognition receptors (PRRs) that recognize essential bacterial molecules, so-called pathogen-associated molecular patterns (PAMPs). A subset of PRRs, belonging to the NOD-like receptor (NLR) and the PYHIN protein families, detects viral and bacterial pathogens in the cytosol of host cells and induces the assembly of a multi-protein signaling platform called the inflammasome. The inflammasome serves as an activation platform for the mammalian cysteine protease caspase-1, a central mediator of innate immunity. Active caspase-1 promotes the maturation and release of interleukin-1β (IL-1β) and IL-18 as well as protein involved in cytoprotection and tissue repair. In addition, caspase-1 initiates a novel form of cell death called pyroptosis. Here, we discuss latest advances and our insights on inflammasome stimulation by two model intracellular pathogens, Francisella tularensis and Salmonella typhimurium. Recent studies on these pathogens have significantly shaped our understanding of the molecular mechanisms of inflammasome activation and how microbes can evade or manipulate inflammasome activity. In addition, we review the role of the inflammasome adapter ASC in caspase-1 autoproteolysis and new insights into the structure of the inflammasome complex.     

Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium

Cullins function as scaffolds that, along with F-box/WD40-repeat-containing proteins, mediate the ubiquitination of proteins to target them for degradation by the proteasome. We have identified a cullin CulA that is required at several stages during Dictyostelium development. culA null cells are defective in inducing cell-type-specific gene expression and exhibit defects during aggregation, including reduced chemotaxis. PKA is an important regulator of Dictyostelium development. The levels of intracellular cAMP and PKA activity are controlled by the rate of synthesis of cAMP and its degradation by the cAMP-specific phosphodiesterase RegA. We show that overexpression of the PKA catalytic subunit (PKAcat) rescues many of the culA null defects and those of cells lacking FbxA/ChtA, a previously described F-box/WD40-repeat-containing protein, suggesting CulA and FbxA proteins are involved in regulating PKA function. Whereas RegA protein levels drop as the multicellular organism forms in the wild-type strain, they remain high in culA null and fbxA null cells. Although PKA can suppress the culA and fbxA null developmental phenotypes, it does not suppress the altered RegA degradation, suggesting that PKA lies downstream of RegA, CulA, and FbxA. Finally, we show that CulA, FbxA, and RegA are found in a complex in vivo, and formation of this complex is dependent on the MAP kinase ERK2, which is also required for PKA function. We propose that CulA and FbxA regulate multicellular development by targeting RegA for degradation via a pathway that requires ERK2 function, leading to an increase in cAMP and PKA activity.     

Cellular inhibitors of apoptosis proteins cIAP1 and cIAP2 are required for efficient caspase-1 activation by the inflammasome

Pathogen and danger recognition by the inflammasome activates inflammatory caspases that mediate inflammation and cell death. The cellular inhibitor of apoptosis proteins (cIAPs) function in apoptosis and innate immunity, but their role in modulating the inflammasome and the inflammatory caspases is unknown. Here we report that the cIAPs are critical effectors of the inflammasome and are required for efficient caspase-1 activation. cIAP1, cIAP2, and the adaptor protein TRAF2 interacted with caspase-1-containing complexes and mediated the activating nondegradative K63-linked polyubiquitination of caspase-1. Deficiency in cIAP1 (encoded by Birc2) or cIAP2 (Birc3) impaired caspase-1 activation after spontaneous or agonist-induced inflammasome assembly, and Birc2(-/-) or Birc3(-/-) mice or mice administered with an IAP antagonist had a dampened response to inflammasome agonists and were resistant to peritonitis. Our results describe a role for the cIAPs in innate immunity and further demonstrate the evolutionary conservation between cell death and inflammation mechanisms.     

Epitope-specific signaling through CD45 on T lymphocytes leads to cAMP synthesis in monocytes after ICAM-1-dependent cellular interaction

We recently demonstrated that different CD45 monoclonal antibodies (mAb) are able to induce cellular aggregation in human peripheral blood mononuclear cells (PBMC) through LFA-1/ICAM-1 interactions. Such interactions could be down-modulated by protein kinase (PK) A/G inhibitors, but were unaffected by inhibitors of PKC, suggesting the involvement of PKA or PKG in CD45 mAb-induced adhesion. In this study we show that after incubation of PBMC with several (but not all) mAb to CD45, CD45RO and CD45RA, intracellular cAMP, but not cGMP concentrations readily increase, reaching a maximum 30 min after start of activation. As evidenced by several lines of investigation cAMP accumulation was independent of Fc receptor-associated signaling as well as tyrosine phosphatase activity of CD45. In highly pure T lymphocytes, CD45 mAb were unable to induce cAMP synthesis, but readily did so after addition of autologous monocytes. After paraformaldehyde fixation of both quiescent or IFN-γ/TNF-α-preactivated monocytes, cAMP production was no longer detectable, suggesting monocytes as the cell of origin for the increased cAMP synthesis. Further, cAMP accumulation in monocytes occurred after reconstitution to T lymphocytes preincubated with CD45 mAb and extensively washed. Importantly, pretreatment of T lymphocyte/monocyte mixtures with LFA-1 mAb and/or ICAM-1 mAb down-regulated CD45 mAb-induced cAMP synthesis. Finally, we demonstrate that CD45 mAb are not only capable of inducing cAMP production, but also of directly stimulating PKA enzyme activity. Based on the data presented, we propose that CD45 signaling in T lymphocytes subsequently activates cAMP accumulation and PKA activation in monocytes via LFA-1/ICAM-1-dependent cellular interactions.     

The cyclic AMP-dependent protein kinase a network regulates development and virulence in Aspergillus fumigatus

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. To determine the importance of the cyclic AMP (cAMP) signaling pathway for virulence, the pkaC1 gene encoding a protein kinase A (PKA) catalytic subunit was cloned and characterized. Deletion of pkaC1 led to reduced conidiation and growth. PKA activity was not detectable in DeltapkaC1, DeltagpaB, and DeltaacyA mutant strains. gpaB and acyA encode a G protein alpha subunit involved in cAMP signal transduction and adenylate cyclase, respectively. Addition of cAMP led to PKA activity in crude extracts of both the DeltagpaB and DeltaacyA strains but not in crude extracts of the DeltapkaC1 strain. These findings provide evidence that PKAC1 represents the predominant form of PKA under the conditions tested, and GPAB and ACYA are members of the cAMP signaling cascade. Analysis of a pksPp-lacZ gene fusion indicated that the expression of the pathogenicity determinant-encoding pksP gene was reduced in DeltapkaC1 mutant strains compared with the expression of the gene fusion in the parental strain. In a low-dose murine inhalation model, conidia of both the DeltapkaC1 and DeltagpaB mutant strains were almost avirulent. Taken together, these findings indicate that the cAMP-PKA signal transduction pathway is required for A. fumigatus pathogenicity.     

Molecular Mechanism of NLRP3 Inflammasome Activation

The inflammasome is an intracellular multimolecular complex that controls caspase-1 activity in the innate immune system. NLRP3, a member of the NLR family of cytosolic pattern recognition receptors, along with the adaptor protein ASC, mediates caspase-1 activation via assembly of the inflammasome in response to various pathogen-derived factors as well as danger-associated molecules. The active NLRP3 inflammasome drives innate immune response towards invading pathogens and cellular damage, and regulates adaptive immune response. Here, we review identified agonists of the NLRP3 inflammasome and the molecular mechanism by which they induce NLRP3 inflammasome activation. Three signaling pathways involving potassium efflux, generation of reactive oxygen species, and cathepsin B release are discussed.     

Cirtical role for Salmonella effector SopB in regulating inflammasome activation

ObjectiveSalmonella is known to evolve many mechanisms to avoid or delay inflammasome activation which remain largely unknown. In this study, we investigated whether the SopB protein critical to bacteria virulence capacity was an effector that involved in the regulation of inflammasome activation.MethodsBMDMs from NLRC4-, NLRP3-, caspase-1/-11-, IFI16- and AIM2-deficient mice were pretreated with LPS, and subsequently stimulated with a series of SopB-related strains of Salmonella, inflammasome induced cell death, IL-1β secretion, cleaved caspase-1 production and ASC speckle formation were detected.ResultsWe found that SopB could inhibit host IL-1β secretion, caspase-1 activation and inflammasome induced cell death using a series of SopB-related strains of Salmonella; however the reduction of IL-1β secretion was not dependent on sensor that contain PYD domain, such as NLRP3, AIM2 or IFI16, but dependent on NLRC4. Notably, SopB specifically prevented ASC oligomerization and the enzymatic activity of SopB was responsible for the inflammasome inhibition. Furthermore, inhibition of Akt signaling induced enhanced inflammasome activation.ConclusionsThese results revealed a novel role in inhibition of NLRC4 inflammasome for Salmonella effector SopB.     

Short communication: differences between macrophages and dendritic cells in the cyclic AMP-dependent regulation of lipopolysaccharide-induced cytokine and chemokine synthesis.

Cyclic adenosine monophosphate (cAMP) is an intracellular signaling molecule responsible for directing cellular responses to extracellular signals. Once believed to signal exclusively through its ability to bind protein kinase A (PKA), recent research has revealed alternative cAMP-binding targets involved in PKA-independent processes. In this study we addressed the hypothesis that the guanine nucleotide exchange protein directly activated by cAMP (Epac-1) and PKA differentially regulate inflammatory mediator production in distinct phagocytic cell types. To accomplish this, we compared the release of cAMP-regulated polypeptide inflammatory mediators in both macrophages (obtained from the lung and peritoneum) and bone marrow-derived dendritic cells (DCs) stimulated with bacterial endotoxin. Using the highly selective Epac-1 and PKA activating cAMP analogs 8-pCPT-2 -O-Me-cAMP and 6-Bnz-cAMP, respectively, we found that macrophages differ from DCs in the involvement of these distinct cAMP pathways in modulating inflammatory mediator release in response to endotoxin. Whereas the regulation of cytokine and chemokine production in macrophages by cAMP was solely dependent on PKA, we found that both Epac-1 and PKA activation could regulate mediator production in DCs. This finding may be important in the pharmacologic regulation of immune responses through manipulation of cAMP signaling cascades and contributes to our understanding of the differences between these cell types.