Oestrus ovis (Diptera: Oestridae): effects of larval excretory/secretory products on nitric oxide production by murine RAW 264.7 macrophages

Larvae of Oestrus ovis (Insecta: Diptera: Oestridae) are common parasites of nasal and sinus cavities of sheep and goats. Previous studies revealed that crude extracts of larvae modify NO synthesis by ovine monocyte derived macrophages. The aim of this study was to investigate the larval excretory/secretory products effects on nitric oxide production by murine tumour macrophages RAW 264.7. Stimulation of RAW macrophages by excretory/secretory products of the three instars larvae (25 microg/ml) significantly increased nitrite concentrations in culture supernatants compared to negative and positive Escherichia coli lipopolysaccharide control. This effect was time and dose dependent. Nitrite production in culture supernatants was due to induction of isoform NOS-2 because both NG monomethyl L-arginine (100 microM) and dexamethasone (20 microM) inhibited, by 60 and 50%, respectively, nitrite accumulation in culture supernatants. First steps of purification, by ion exchange chromatography, indicated that one protein of 29 kDa was able to induce NO synthesis by macrophages. Further studies are needed for a better characterization of these molecule and to investigate their immunogenicity for a vaccine approach.     

Regulation of TLR4 signaling through the TRAF6/sNASP axis by reversible phosphorylation mediated by CK2 and PP4

Recognition of invading pathogens by Toll-like receptors (TLRs) activates innate immunity through signaling pathways that involved multiple protein kinases and phosphatases. We previously demonstrated that somatic nuclear autoantigenic sperm protein (sNASP) binds to TNF receptor–associated factor 6 (TRAF6) in the resting state. Upon TLR4 activation, a signaling complex consisting of TRAF6, sNASP, interleukin (IL)-1 receptor–associated kinase 4, and casein kinase 2 (CK2) is formed. CK2 then phosphorylates sNASP to release phospho-sNASP (p-sNASP) from TRAF6, initiating downstream signaling pathways. Here, we showed that protein phosphatase 4 (PP4) is the specific sNASP phosphatase that negatively regulates TLR4-induced TRAF6 activation and its downstream signaling pathway. Mechanistically, PP4 is directly recruited by phosphorylated sNASP to dephosphorylate p-sNASP to terminate TRAF6 activation. Ectopic expression of PP4 specifically inhibited sNASP-dependent proinflammatory cytokine production and downstream signaling following bacterial lipopolysaccharide (LPS) treatment, whereas silencing PP4 had the opposite effect. Primary macrophages and mice infected with recombinant adenovirus carrying a gene encoding PP4 (Ad-PP4) showed significant reduction in IL-6 and TNF-α production. Survival of Ad-PP4–infected mice was markedly increased due to a better ability to clear bacteria in a sepsis model. These results indicate that the serine/threonine phosphatase PP4 functions as a negative regulator of innate immunity by regulating the binding of sNASP to TRAF6.

Toll-like receptors (TLRs) are crucial for initiating the innate immune response and are initiated by recognizing pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide (LPS) (1–3). Upon detection of PAMPs, most TLRs (except TLR3), associate with the adaptor protein MyD88, which initiates the signaling process through interleukin 1 receptor–associated kinases and subsequently promotes the auto-ubiquitination of TNF receptor–associated factor 6 (TRAF6). Ubiquitinated TRAF6 then transmits intracellular signals through phosphorylation of transforming growth factor β-activated protein kinase 1 (TAK1), which results in the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) and subsequent production of a wide range of immune stimulatory cytokines and chemokines to aid against invading microorganisms (1, 4, 5). Dysregulation of TLRs signaling was implicated in human diseases, including sepsis, autoimmune diseases, cancer, and pulmonary fibrosis (6–13). Thus, tight regulation of TLRs signaling is necessary for homeostasis of an appropriate immune response.Reversible protein phosphorylation and dephosphorylation serve as regulatory switches by regulating the activation and deactivation of multiple TLR-dependent signaling molecules, such as extracellular signal–related kinase (ERK), c-jun N-terminal kinase (JNK), p38α MAPK, IκB kinase (IKK), and interferon regulatory factor 3 (IRF3) (4, 14). The role of kinases in regulating the response of macrophages to TLR signaling has been extensively studied. Phosphorylation of both p38α and JNK directly mediated by TAK1 is critical for TLR-induced MAPK activation (15). The NF-κB subunit precursor protein p105 undergoes phosphorylation, which is necessary for K48-linked ubiquitylation and proteasome-mediated proteolysis of the IKK complex (16). Moreover, casein kinase 2 (CK2) was found to phosphorylate IκBα and the p65 unit to regulate NF-κB–mediated inflammation (17, 18). Our previous study has also indicated that CK2-mediated phosphorylation of somatic nuclear autoantigenic sperm protein (sNASP) is a key element activating TRAF6 downstream signaling during LPS stimulation, but it is still unknown how the sNASP/TRAF6-mediated pathway is negatively regulated (19).Many proteins were identified that limit TLR signaling through regulating protein dephosphorylation, such as SH2-containing protein tyrosine phosphatase 2 (SHP-2) (20), NLR family member X1 (21), NLRP6 (22), and protein phosphatase 2A (PP2A) (23). As an example, PP2A suppresses TLRs-triggered type I IFN signaling by deactivation of IRF3 via dephosphorylation. Furthermore, PP2A-deficient macrophages showed enhanced type I IFN signaling upon stomatitis virus infection (23). SHP-2 negatively regulated TLR4- and TLR3-activated IFN-β and production of proinflammatory cytokines, interleukin (IL)-6 and TNF-α, by directly binding TANK binding kinase (20). Here, we report that the serine and threonine protein phosphatase 4 (PP4) is the phosphatase for phosphorylated sNASP and that after TLR4 stimulation, phospho-sNASP (p-sNASP) is dephosphorylated to return to a resting state. Our results also suggested that dephosphorylation of sNASP plays an essential role in the regulation of the LPS signaling cascade. The interaction between sNASP and PP4 is mediated by phosphorylation of sNASP by CK2. The phosphorylated sNASP/PP4 complex was found to transiently form upon LPS challenge. In vivo, PP4 overexpression alleviates the expression of proinflammatory cytokine in macrophages and protects mice from endotoxic shock. Thus, we present a critical role of PP4 in TLR signaling that targets phosphorylated sNASP, which contributes to homeostasis of the TLR/sNASP/TRAF6 axis in the innate immune response.PP4 is a serine/threonine protein phosphatase that is comprised of a catalytic subunit (PP4C) and regulatory subunits PP4R (24, 25). PP4 has been highly conserved during eukaryote evolution, suggesting that PP4, like PP2A and PP6, might have critical functions in vivo (26). Although previous studies showed that PP4 was implicated in many cellular processes, including organelle assembly (27, 28), DNA damage repair (29), embryo development (30), pro-B-cell development (31), and regulatory T cell functions (32), its roles in regulation of proinflammatory signaling and innate immunity have not been demonstrated. In the present study, we identified serine/threonine phosphatase PP4 as a specific phosphatase for sNASP that is essential for the termination of TRAF6 activation. This knowledge may help in the developing therapeutic strategies for deregulating proinflammatory cytokine.     

Differential IL-4/Stat6 activities correlate with differential expression of regulatory genes SOCS-1 , SHP-1 , and PP2A in colon cancer cells

Purpose  To investigate potential differences in the expression of Stat6 regulatory genes that may influence IL-4/Stat6 activities (phenotypes) in colon cancer cells. Methods  RT-PCR method was employed to examine the constitutive mRNA expression of Stat6 negative regulators SOCS-1 and SHP-1, and positive regulator PP2A in colon cancer cell lines HT-29 and Caco-2. Stat6 protein expression and nuclear phosphorylation were detected using Western blotting. Results  Caco-2 cells carrying inactive Stat6^null phenotype showed normal constitutive expression of Stat6 but decreased phosphorylation of nuclear Stat6 compared with HT-29 cells carrying active Stat6^high phenotype. Stat6^null Caco-2 cells expressed increased levels of mRNA and protein of SOCS-1 and SHP-1, and decreased mRNA expression of PPP2CA and PPP2CB, encoding two critical subunits of PP2A. Conclusions  Constitutively increased expression of Stat6 negative regulators SOCS-1 and SHP-1, together with decreased expression of positive regulator PP2A, may play a role in forming the inactive Stat6^null phenotype in colon cancer cells. Qin Yuan and Pin Dong Li contributed equally to this work.     

Cytokine control of Leishmania infection in the BALB/c mouse: enhancement and inhibition of parasite growth by local administration of IL-2 or IL-4 is species and time dependent

The therapeutic potential of locally injected interleukin-2 (IL-2) or interleukin-4 (IL-4) was studied in the footpads of Leishmania mexicana or Leishmania major infected BALB/c mice. The disease state was measured both pathologically, by measuring lesion size, and parasitologically, by counting total parasite numbers from infected footpads. IL-2 (0.5 microgram/dose) or IL-4 (0.1 microgram/dose) was administered either early, 1 day and/or 15 days after infection, or late, after palpable lesions had developed. Results differed markedly depending on which Leishmania species was used and at what time during the course of disease that therapy commenced. Both L. major and L. mexicana infections, as measured by footpad thickness and parasite number, were exacerbated if IL-4 was injected into the infected footpads early, during the first two weeks of infection. Paradoxically, late intralesional injection (i.e. after measurable lesions had developed) of IL-4 markedly inhibited both lesion size and parasite growth in L. major, though not L. mexicana, infected mice. IL-2 had no measurable effect on the course of L. major infections no matter when or how often, the infected footpads of mice were treated. However, early administration of IL-2 did exacerbate L. mexicana lesion and parasite growth while late treatment had no effect. Generally, but not always, increases in footpad size correlated with increases in parasite number.     

Cyclic GMP/PKG-dependent inhibition of TRPC6 channel activity and expression negatively regulates cardiomyocyte NFAT activation: Novel mechanism of cardiac stress modulation by PDE5 inhibition

Increased cyclic GMP from enhanced synthesis or suppressed catabolism (e.g. PDE5 inhibition by sildenafil, SIL) activates protein kinase G (PKG) and blunts cardiac pathological hypertrophy. Suppressed calcineurin (Cn)-NFAT (nuclear factor of activated T-cells) signaling appears to be involved, though it remains unclear how this is achieved. One potential mechanism involves activation of Cn/NFAT by calcium entering via transient receptor potential canonical (TRPC) channels (notably TRPC6). Here, we tested the hypothesis that PKG blocks Cn/NFAT activation by modifying and thus inhibiting TRPC6 current to break the positive feedback loop involving NFAT and NFAT-dependent TRPC6 upregulation. TRPC6 expression rose with pressure-overload in vivo, and angiotensin (ATII) or endothelin (ET1) stimulation in neonatal and adult cardiomyocytes in vitro. 8Br-cGMP and SIL reduced ET1-stimulated TRPC6 expression and NFAT dephosphorylation (activity). TRPC6 upregulation was absent if its promoter was mutated with non-functional NFAT binding sites, whereas constitutively active NFAT triggered TRPC6 expression that was not inhibited by SIL. PKG phosphorylated TRPC6, and both T70 and S322 were targeted. Both sites were functionally relevant, as 8Br-cGMP strongly suppressed current in wild-type TRPC6 channels, but not in those with phospho-silencing mutations (T70A, S322A or S322Q). NFAT activation and increased protein synthesis stimulated by ATII or ET1 was blocked by 8Br-cGMP or SIL. However, transfection with T70A or S322Q TRPC6 mutants blocked this inhibitory effect, whereas phospho-mimetic mutants (T70E, S322E, and both combined) suppressed NFAT activation. Thus PDE5-inhibition blocks TRPC6 channel activation and associated Cn/NFAT activation signaling by PKG-dependent channel phosphorylation.     

Differential involvement of signaling pathways in the regulation of growth hormone release by somatostatin and growth hormone-releasing hormone in orange-spotted grouper (Epinephelus coioides)

Somatostatin is the most effective inhibitor of GH release, and GHRH was recently identified as one of the primary GH-releasing factors in teleosts. In this study, we analyzed the possible intracellular transduction pathways that are involved in the mechanisms induced by SRIF and GHRH to regulate GH release. Using a pharmacological approach, the blockade of the PLC/IP/PKC pathway reversed the SRIF-induced inhibition of GH release but did not affect the GHRH-induced stimulation of GH release. Furthermore, SRIF reduced the GH release induced by two PKC activators. Inhibitors of the AC/cAMP/PKA pathway reversed both the SRIF- and GHRH-induced effects on GH release. Moreover, the GH release evoked by forskolin and 8-Br-cAMP were completely abolished by SRIF. The blockade of the NOS/NO pathway attenuated the GHRH-induced GH release but had minimal effects on the inhibitory actions of SRIF. In addition, inhibitors of the sGC/cGMP pathway did not modify the SRIF- or GHRH-induced regulation of GH release. Taken together, these findings indicate that the SRIF-induced inhibition of GH release is mediated by both the PLC/IP/PKC and the AC/cAMP/PKA pathways and not by the NOS/NO/sGC/cGMP pathway. In contrast, the GHRH-induced stimulation of GH secretion is mediated by both the AC/cAMP/PKA and the NOS/NO pathways and is independent of the sGC/cGMP pathway and the PLC/IP/PKC system.     

The role of STAT, AP-1, E-box and TIEG motifs in the regulation of hepcidin by IL-6 and BMP-9: lessons from human HAMP and murine Hamp1 and Hamp2 gene promoters

Hepcidin, the principal regulator of the iron metabolism, is up-regulated in response to inflammatory stimuli, bone morphogenic proteins (BMPs) and iron excess. There are two murine hepcidin genes: hepcidin-1 (Hamp1) and hepcidin-2 (Hamp2). Hamp1 gene responds to both IL-6 and BMPs while Hamp2 responds to neither. We replaced the putative functional regulatory motifs of the Hamp1 promoter with the corresponding putative "non-functional" Hamp2 motifs and vice versa in reporter constructs. Conversion of the Hamp1 STAT site into the Hamp2 site reduced the basal level of reporter expression but did not affect IL-6 and BMP responsiveness; replacing Hamp2 site with the Hamp1 site only resulted in partial responsiveness. These data are in contrast to the role of the STAT site in the human hepcidin promoter which is important in both basal level and IL-6 inducible promoter activity. The murine AP1, E-box and TIEG motifs were found to neither influence the basal level of expression of Hamp1 and HAMP promoters nor play a critical role in the IL-6 and BMP-9 induced response. Our data suggest that the STAT site (nt -148 to -130) is important for the regulation of basal level expression of Hamp1 but there are additional regions that are responsible for the IL-6 and BMP-9 responsiveness within the Hamp1 promoter.