Differential Arabinan Capping of Lipoarabinomannan Modulates Innate Immune Responses and Impacts T Helper Cell Differentiation

Toll-like receptors (TLRs) recognize pathogens by interacting with pathogen-associated molecular patterns, such as the phosphatidylinositol-based lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM). Such structures are present in several pathogens, including Mycobacterium tuberculosis, being important for the initiation of immune responses. It is well established that the interaction of LM and LAM with TLR2 is a process dependent on the structure of the ligands. However, the implications of structural variations on TLR2 ligands for the development of T helper (Th) cell responses or in the context of in vivo responses are less studied. Herein, we used Corynebacterium glutamicum as a source of lipoglycan intermediates for host interaction studies. In this study, we have deleted a putative glycosyltransferase, NCgl2096, from C. glutamicum and found that it encodes for a novel α(1→2)arabinofuranosyltransferase, AftE. Biochemical analysis of the lipoglycans obtained in the presence (wild type) or absence of NCgl2096 showed that AftE is involved in the biosynthesis of singular arabinans of LAM. In its absence, the resulting molecule is a hypermannosylated (hLM) form of LAM. Both LAM and hLM were recognized by dendritic cells, mainly via TLR2, and triggered the production of several cytokines. hLM was a stronger stimulus for in vitro cytokine production and, as a result, a more potent inducer of Th17 responses. In vivo data confirmed hLM as a stronger inducer of cytokine responses and suggested the involvement of pattern recognition receptors other than TLR2 as sensors for lipoglycans.     

Distinct protein kinase A anchoring proteins direct prostaglandin E2 modulation of Toll-like receptor signaling in alveolar macrophages

Toll-like receptors (TLRs) direct a proinflammatory program in macrophages. One mediator whose generation is induced by TLR ligation is prostaglandin E(2) (PGE(2)), which is well known to increase intracellular cAMP upon G protein-coupled receptor ligation. How PGE(2)/cAMP shapes the nascent TLR response and the mechanisms by which it acts remain poorly understood. Here we explored PGE(2)/cAMP regulation of NO production in primary rat alveolar macrophages stimulated with the TLR4 ligand LPS. Endogenous PGE(2) synthesis accounted for nearly half of the increment in NO production in response to LPS. The enhancing effect of PGE(2) on LPS-stimulated NO was mediated via cAMP, generated mainly upon ligation of the E prostanoid 2 receptor and acting via protein kinase A (PKA) rather than via the exchange protein activated by cAMP. Isoenzyme-selective cAMP agonists and peptide disruptors of protein kinase A anchoring proteins (AKAPs) implicated PKA regulatory subunit type I (RI) interacting with an AKAP in this process. Gene knockdown of potential RI-interacting AKAPs expressed in alveolar macrophages revealed that AKAP10 was required for PGE(2) potentiation of LPS-induced NO synthesis. AKAP10 also mediated PGE(2) potentiation of the expression of cytokines IL-10 and IL-6, whereas PGE(2) suppression of TNF-α was mediated by AKAP8-anchored PKA-RII. Our data illustrate the pleiotropic manner in which G protein-coupled receptor-derived cAMP signaling can influence TLR responses in primary macrophages and suggest that AKAP10 may coordinate increases in gene expression.     

Dendritic cell activation prevents MHC class II ubiquitination and promotes MHC class II survival regardless of the activation stimulus

The expression of MHC class II (MHC-II) on the surface of antigen-presenting cells, such as dendritic cells (DCs), is tightly regulated during cellular activation. Many cells, including DCs, are activated following stimulation of innate Toll-like receptors (TLRs) by products of microorganisms. In the resting (immature) state, MHC-II is ubiquitinated in immature DCs and is rapidly degraded; however, after activation of these cells with MyD88-dependent TLR ligands, MHC-II ubiquitination is blocked, and MHC-II survival is prolonged. We now show that DC activation using MyD88-dependent TLR ligands, MyD88-independent TLR ligands, and even infection with the intracellular parasite Toxoplasma gondii leads to identical changes in MHC-II expression, ubiquitination, and surface stability, revealing a conserved role for enhanced MHC-II stability after DC activation by different stimuli.     

Reengineering the Collision Coupling and Diffusion Mode of the A2A-adenosine Receptor: PALMITOYLATION IN HELIX 8 RELIEVES CONFINEMENT*

The A_2A-adenosine receptor undergoes restricted collision coupling with its cognate G protein G_s and lacks a palmitoylation site at the end of helix 8 in its intracellular C terminus. We explored the hypothesis that there was a causal link between the absence of a palmitoyl moiety and restricted collision coupling by introducing a palmitoylation site. The resulting mutant A_2A-R309C receptor underwent palmitoylation as verified by both mass spectrometry and metabolic labeling. In contrast to the wild type A_2A receptor, the concentration-response curve for agonist-induced cAMP accumulation was shifted to the left with increasing expression levels of A_2A-R309C receptor, an observation consistent with collision coupling. Single particle tracking of quantum dot-labeled receptors confirmed that wild type and mutant A_2A receptor differed in diffusivity and diffusion mode; agonist activation resulted in a decline in mean square displacement of both receptors, but the drop was substantially more pronounced for the wild type receptor. In addition, in the agonist-bound state, the wild type receptor was frequently subject to confinement events (estimated radius 110 nm). These were rarely seen with the palmitoylated A_2A-R309C receptor, the preferred diffusion mode of which was a random walk in both the basal and the agonist-activated state. Taken together, the observations link restricted collision coupling to diffusion limits imposed by the absence of a palmitoyl moiety in the C terminus of the A_2A receptor. The experiments allowed for visualizing local confinement of an agonist-activated G protein-coupled receptor in an area consistent with the dimensions of a lipid raft.     

The extracellular cAMP-adenosine pathway regulates expression of renal D1 dopamine receptors in diabetic rats

Activation of D1 dopamine receptors expressed in the kidneys promotes the excretion of sodium and regulates sodium levels during increases in dietary sodium intake. A decrease in the expression or function of D1 receptors results in increased sodium retention which can potentially lead to the development of hypertension. Studies have shown that in the absence of functional D1 receptors, in null mice, the systolic, diastolic, and mean arterial pressures are higher. Previous studies have shown that the expression and function of D1 receptors in the kidneys are decreased in animal models of diabetes. The mechanisms that down-regulate the expression of renal D1 receptor gene in diabetes are not well understood. Using primary renal cells and acutely isolated kidneys from the streptozotocin-induced rat diabetic model, we demonstrate that the renal D1 receptor expression is down-regulated by the extracellular cAMP-adenosine pathway in vitro and in vivo. In cultures of primary renal cells, a 3 mm, 60-h cAMP treatment down-regulated the expression of D1 receptors. In vivo, we determined that the plasma and urine cAMP levels as well as the expression of 5'-ectonucleotidase, tissue-nonspecific alkaline phosphatase, and adenosine A2a receptors are significantly increased in diabetic rats. Inhibitors of 5'-ectonucleotidase and tissue-nonspecific alkaline phosphatase, α,β-methyleneadenosine 5'-diphosphate, and levamisole, respectively, blocked the down-regulation of D1 receptors in the primary renal cells and in the kidney of diabetic animals. The results suggest that inhibitors of the extracellular cAMP-adenosine pathway reverse the down-regulation of renal D1 receptor in diabetes.     

Characterization of sparstolonin B, a Chinese herb-derived compound, as a selective Toll-like receptor antagonist with potent anti-inflammatory properties

Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2- and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IκBα, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-κB activation, SsnB effectively blunted the NF-κB activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases.     

Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1β secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1β in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1β was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1β and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1β by antigen-presenting cells in an NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1β in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1β through at least two separate mechanisms: by targeting pro-IL-1β for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.     

Raftlin is involved in the nucleocapture complex to induce poly(I:C)-mediated TLR3 activation

The double-stranded RNA analog, poly(I:C), extracellularly activates both the endosomal Toll-like receptor (TLR) 3 and the cytoplasmic RNA helicase, melanoma differentiation-associated gene 5, leading to the production of type I interferons (IFNs) and inflammatory cytokines. The mechanism by which extracellular poly(I:C) is delivered to TLR3-positive organelles and the cytoplasm remains to be elucidated. Here, we show that the cytoplasmic lipid raft protein, Raftlin, is essential for poly(I:C) cellular uptake in human myeloid dendritic cells and epithelial cells. When Raftlin was silenced, poly(I:C) failed to enter cells and induction of IFN-β production was inhibited. In addition, cellular uptake of B-type oligodeoxynucleotide that shares its uptake receptor with poly(I:C) was suppressed in Raftlin knockdown cells. Upon poly(I:C) stimulation, Raftlin was translocated from the cytoplasm to the plasma membrane where it colocalized with poly(I:C), and thereafter moved to TLR3-positive endosomes. Thus, Raftlin cooperates with the uptake receptor to mediate cell entry of poly(I:C), which is critical for activation of TLR3.     

Oral Administration of High Molecular Weight Hyaluronan (900 kDa) Controls Immune System via Toll-like Receptor 4 in the Intestinal Epithelium

Low molecular weight hyaluronan enhances or induces inflammation through toll-like receptor 4 (TLR-4).However, the effects of high molecular weight hyaluronan (HA900) on TLR-4 are unknown. In this study, HA900 (900 kDa) was administered orally to MRL-lpr/lpr mice, a Th-1-type autoimmune disease model. Lymphoaccumulation of double-negative T cells, which is enhanced by proinflammatory cytokines, was suppressed by HA900 treatment. Cytokine array analysis showed that HA900 treatment enhanced production of interleukin-10, an anti-inflammatory cytokine, and down-regulated chemokine production. HA900 colocalized with TLR-4 on the luminal surface of epithelial cells in the large intestine. These cells are parts of the immune system and express cytokines. DNA array analysis of the tissue from the large intestine showed that HA900 treatment up-regulated suppressor of cytokine signaling 3 (SOCS3) expression and down-regulated pleiotrophin expression. Treatment of cultured double-negative T cells from MRL-lpr/lpr mice with pleiotrophin rescued these cells. SOCS3, which is known to suppress inflammation, was enhanced by HA900 treatment. In TLR-4 knockdown HT29 cells (a cell line derived from large intestinal cells), HA900 did not bind to HT29 cells and did not up-regulate SOCS3 expression. Our results suggest that oral administration of HA900 modulates Th-1-type autoimmune disease and inflammation by up-regulating SOCS3 expression and down-regulating pleiotrophin expression via TLR-4 in intestinal epithelial cells.     

Microglial Toll-like receptor 2 contributes to kainic acid-induced glial activation and hippocampal neuronal cell death

Recent studies indicate that Toll-like receptors (TLRs), originally identified as infectious agent receptors, also mediate sterile inflammatory responses during tissue damage. In this study, we investigated the role of TLR2 in excitotoxic hippocampal cell death using TLR2 knock-out (KO) mice. TLR2 expression was up-regulated in microglia in the ipsilateral hippocampus of kainic acid (KA)-injected mice. KA-mediated hippocampal cell death was significantly reduced in TLR2 KO mice compared with wild-type (WT) mice. Similarly, KA-induced glial activation and proinflammatory gene expression in the hippocampus were compromised in TLR2 KO mice. In addition, neurons in organotypic hippocampal slice cultures (OHSCs) from TLR2 KO mouse brains were less susceptible to KA excitotoxicity than WT OHSCs. This protection is partly attributed to decreased expression of proinflammatory genes, such as TNF-α and IL-1β in TLR2 KO mice OHSCs. These data demonstrate conclusively that TLR2 signaling in microglia contributes to KA-mediated innate immune responses and hippocampal excitotoxicity.     

Profibrotic Effect of Interleukin-18 in HK-2 Cells Is Dependent on Stimulation of the Toll-like Receptor 4 (TLR4) Promoter and Increased TLR4 Expression

IL-18 is an important mediator of obstruction-induced renal fibrosis and tubular epithelial cell injury independent of TGF-β1 activity. We sought to determine whether the profibrotic effect of IL-18 is mediated through Toll-like receptor 4 (TLR4). Male C57BL6 wild type and mice transgenic for human IL-18-binding protein were subjected to left unilateral ureteral obstruction versus sham operation. The kidneys were harvested 1 week postoperatively and analyzed for IL-18 production and TLR4 expression. In a separate arm, renal tubular epithelial cells (HK-2) were directly stimulated with IL-18 in the presence or absence of a TLR4 agonist, TLR4 antagonist, or TLR4 siRNA knockdown. Cell lysates were analyzed for TLR4, α-smooth muscle actin, and E-cadherin expression. TLR4 promotor activity, as well as AP-1 activation and the effect of AP-1 knockdown on TLR4 expression, was evaluated in HK-2 cells in response to IL-18 stimulation. The results demonstrate that IL-18 induces TLR4 expression during unilateral ureteral obstruction and induces TLR4 expression in HK-2 cells via AP-1 activation. Inhibition of TLR4 or knockdown of TLR4 gene expression in turn prevents IL-18-induced profibrotic changes in HK-2 cells. These results suggest that IL-18 induces profibrotic changes in tubular epithelial cells via increased TLR4 expression/signaling.     

Regulation of oocyte and cumulus cell interactions by intermedin/adrenomedullin 2

Ovarian folliculogenesis has been studied as a model of hormonal regulation of development and differentiation, cell death, and cell-cell communication. In addition to gonadotropins from the pituitary and follicular paracrine factors, oocyte secreted factors have been shown to play critical roles in the regulation of follicular cell functions. Except for the well characterized BMP family proteins, including GDF9 and BMP15, oocytes are known to secrete oocyte secreted factors that are important for the regulation of cumulus cell survival and the maintenance of tertiary structure of cumulus cell-enclosed oocyte complexes (COCs). Based on genomic screening and studies of COCs cultured in vitro, we showed that intermedin (IMD)/adrenomedullin 2 (ADM2) is a novel oocyte-derived ligand important for the regulation of cell interactions in COCs that functions, in part, by suppressing cumulus cell apoptosis. Consistently, we showed that suppression of IMD/ADM2 signaling in growing rat ovaries in vivo leads to oocyte atresia and aberrant cell cycle progression in follicular cells. Together, our studies indicated that mammalian oocytes deploy a G protein-coupled receptor ligand to coordinate normal interactions of oocytes and cumulus cells and provided a better understanding of how the tertiary structure of a COC is maintained as follicles undergo exponential growth during the late stages of folliculogenesis.     

Pivotal role of ADP-ribosylation factor 6 in Toll-like receptor 9-mediated immune signaling

CpG oligodeoxynucleotide (CpG ODN) cellular uptake into endosomes, the rate-limiting step of Toll-like receptor 9 (TLR9) signaling, is critical in eliciting innate immune responses. ADP-ribosylation factor 6 (ARF6) is a member of the Ras superfamily, which is critical to a wide variety of cellular events including endocytosis. Here, we found that inhibition of ARF6 by dominant mutants and siRNA impaired CpG ODN-mediated responses, whereas cells expressing the constitutively active ARF6 mutant enhanced CpG ODN-induced cytokine production. Inhibition of ARF6 impaired TLR9 trafficking into endolysosomes, thereby inhibiting proceed functional cleavage of TLR9. Additional studies showed that CpG ODN uptake was increased in ARF6-activated cells but impaired in ARF6-defective cells. Furthermore, cells pretreated with CpG ODN but not GpC ODN had increased CpG ODN uptake due to CpG ODN-induced ARF6 activity. Further studies with ARF6-defective and ARF6-activated cells demonstrated that class III phosphatidylinositol 3-kinases (PI3K) was required for downstream ARF6 regulation of CpG ODN uptake. Together, our findings demonstrate that a novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN.     

Mycobacterium paratuberculosis CobT Activates Dendritic Cells via Engagement of Toll-like Receptor 4 Resulting in Th1 Cell Expansion

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne disease in animals and MAP involvement in human Crohn disease has been recently emphasized. Evidence from M. tuberculosis studies suggests mycobacterial proteins activate dendritic cells (DCs) via Toll-like receptor (TLR) 4, eventually determining the fate of immune responses. Here, we investigated whether MAP CobT contributes to the development of T cell immunity through the activation of DCs. MAP CobT recognizes TLR4, and induces DC maturation and activation via the MyD88 and TRIF signaling cascades, which are followed by MAP kinases and NF-κB. We further found that MAP CobT-treated DCs activated naive T cells, effectively polarized CD4⁺ and CD8⁺ T cells to secrete IFN-γ and IL-2, but not IL-4 and IL-10, and induced T cell proliferation. These data indicate that MAP CobT contributes to T helper (Th) 1 polarization of the immune response. MAP CobT-treated DCs specifically induced the expansion of CD4⁺/CD8⁺CD44^highCD62L^low memory T cells in the mesenteric lymph node of MAP-infected mice in a TLR4-dependent manner. Our results indicate that MAP CobT is a novel DC maturation-inducing antigen that drives Th1 polarized-naive/memory T cell expansion in a TLR4-dependent cascade, suggesting that MAP CobT potentially links innate and adaptive immunity against MAP.     

Osteoblastic γ-aminobutyric acid, type B receptors negatively regulate osteoblastogenesis toward disturbance of osteoclastogenesis mediated by receptor activator of nuclear factor κB ligand in mouse bone

The prevailing view is that signaling machineries for the neurotransmitter GABA are also expressed by cells outside the CNS. In cultured murine calvarial osteoblasts, mRNA was constitutively expressed for both subunits 1 and 2 of metabotropic GABA(B) receptor (GABA(B)R), along with inhibition by the GABA(B)R agonist baclofen of cAMP formation, alkaline phosphatase (ALP) activity, and Ca(2+) accumulation. Moreover, baclofen significantly inhibited the transactivation of receptor activator of nuclear factor-κB ligand (RANKL) gene in a manner sensitive to a GABA(B)R antagonist, in addition to decreasing mRNA expression of bone morphogenetic protein-2 (BMP2), osteocalcin, and osterix. In osteoblastic MC3T3-E1 cells stably transfected with GABA(B)R1 subunit, significant reductions were seen in ALP activity and Ca(2+) accumulation, as well as mRNA expression of osteocalcin, osteopontin, and osterix. In cultured calvarial osteoblasts from GABA(B)R1-null mice exhibiting low bone mineral density in tibia and femur, by contrast, both ALP activity and Ca(2+) accumulation were significantly increased together with promoted expression of both mRNA and proteins for BMP2 and osterix. No significant change was seen in the number of multinucleated cells stained for tartrate-resistant acid phosphatase during the culture of osteoclasts prepared from GABA(B)R1-null mice, whereas a significant increase was seen in the number of tartrate-resistant acid phosphatase-positive multinucleated cells in co-culture of osteoclasts with osteoblasts isolated from GABA(B)R1-null mice. These results suggest that GABA(B)R is predominantly expressed by osteoblasts to negatively regulate osteoblastogenesis through down-regulation of BMP2 expression toward disturbance of osteoclastogenesis after down-regulation of RANKL expression in mouse bone.     

WAVE2 forms a complex with PKA and is involved in PKA enhancement of membrane protrusions

PKA contributes to many physiological processes, including glucose homeostasis and cell migration. The substrate specificity of PKA is low compared with other kinases; thus, complex formation with A-kinase-anchoring proteins is important for the localization of PKA in specific subcellular regions and the phosphorylation of specific substrates. Here, we show that PKA forms a complex with WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) in MDA-MB-231 breast cancer cells and mouse brain extracts. Two separate regions of WAVE2 are involved in WAVE2-PKA complex formation. This complex localizes to the leading edge of MDA-MB-231 cells. PKA activation results in enlargement of the membrane protrusion. WAVE2 depletion impairs PKA localization at membrane protrusions and the enlargement of membrane protrusion induced by PKA activation. Together, these results suggest that WAVE2 works as an A-kinase-anchoring protein that recruits PKA at membrane protrusions and plays a role in the enlargement of membrane protrusions induced by PKA activation.     

Adenosine A2A Receptor Signaling and Golf Assembly Show a Specific Requirement for the γ7 Subtype in the Striatum

The adenosine A_2A receptor (A_2AR) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α_olf subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ₇ subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ₇ protein controls the hierarchial assembly of a specific G-protein α_olfβ₂γ₇ heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A_2AR activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ₇ subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A_2AR G-protein α_olfβ₂γ₇ interface as a possible therapeutic target for Parkinson disease.