Toll-like receptor 4 signalling pathway activation in a rat model of Acanthamoeba Keratitis

The pathogenesis of Acanthamoeba keratitis (AK) is complicated. In our previous studies, TLR4 was found involved in the process of infection by Acanthamoeba in human corneal cells. The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) signalling pathway in Wistar rats challenged with Acanthamoeba. The rat model of AK was established. Corneas were collected and analysed by real-time PCR to assess the mRNA levels of TLR 2, 4, myeloid differentiation protein (MyD)88, nuclear factor (NF)-κB, extracellular signal-regulated kinase (ERK), interleukin (IL)-8, tumour necrosis factor (TNF)-α and interferon (IFN) -β. Immunocytochemistry and Western blot were conducted to examine the proteins of TLR2, TLR4, p-Erk1/2 and p-IκB. Specific inhibitors PDTC and U0126 were used to pretreat the animals to determine the exact receptor and signalling pathway involved in pathogenesis. Expressions of TLR4, MyD88, all three cytokines, NF-κB, p-IκB and p-Erk1/2 were increased in Acanthamoeba-treated rat corneas. PDTC inhibited the production of IL-8 and TNF-α, while U0126 inhibited the synthesis of IFN-β. TLR4 was involved in sensing the challenge of Acanthamoeba and inducing production of cytokines through TLR4-NF-κB and TLR4-Erk1/2 pathways in corneas of Wistar rats.     

Brain angiogenesis inhibitor 1 (BAI1) is a pattern recognition receptor that mediates macrophage binding and engulfment of Gram-negative bacteria

Bacterial recognition by host cells is essential for initiation of infection and the host response. Bacteria interact with host cells via multiple pattern recognition receptors that recognize microbial products or pathogen-associated molecular patterns. In response to this interaction, host cell signaling cascades are activated that lead to inflammatory responses and/or phagocytic clearance of attached bacteria. Brain angiogenesis inhibitor 1 (BAI1) is a receptor that recognizes apoptotic cells through its conserved type I thrombospondin repeats and triggers their engulfment through an ELMO1/Dock/Rac1 signaling module. Because thrombospondin repeats in other proteins have been shown to bind bacterial surface components, we hypothesized that BAI1 may also mediate the recognition and clearance of pathogenic bacteria. We found that preincubation of bacteria with recombinant soluble BAI1 ectodomain or knockdown of endogenous BAI1 in primary macrophages significantly reduced binding and internalization of the Gram-negative pathogen Salmonella typhimurium. Conversely, overexpression of BAI1 enhanced attachment and engulfment of Salmonella in macrophages and in heterologous nonphagocytic cells. Bacterial uptake is triggered by the BAI1-mediated activation of Rac through an ELMO/Dock-dependent mechanism, and inhibition of the BAI1/ELMO1 interaction prevents both Rac activation and bacterial uptake. Moreover, inhibition of ELMO1 or Rac function significantly impairs the proinflammatory response to infection. Finally, we show that BAI1 interacts with a variety of Gram-negative, but not Gram-positive, bacteria through recognition of their surface lipopolysaccharide. Together these findings identify BAI1 as a pattern recognition receptor that mediates nonopsonic phagocytosis of Gram-negative bacteria by macrophages and directly affects the host response to infection.     

RoR2 functions as a noncanonical Wnt receptor that regulates NMDAR-mediated synaptic transmission

Wnt signaling has a well-established role as a regulator of nervous system development, but its role in the maintenance and regulation of established synapses in the mature brain remains poorly understood. At excitatory glutamatergic synapses, NMDA receptors (NMDARs) have a fundamental role in synaptogenesis, synaptic plasticity, and learning and memory; however, it is not known what controls their number and subunit composition. Here we show that the receptor tyrosine kinase-like orphan receptor 2 (RoR2) functions as a Wnt receptor required to maintain basal NMDAR-mediated synaptic transmission. In addition, RoR2 activation by a noncanonical Wnt ligand activates PKC and JNK and acutely enhances NMDAR synaptic responses. Regulation of a key component of glutamatergic synapses through RoR2 provides a mechanism for Wnt signaling to modulate synaptic transmission, synaptic plasticity, and brain function acutely beyond embryonic development.Wnt ligands are highly conserved secreted glycoproteins responsible for important developmental and homeostatic processes throughout the animal kingdom (1, 2). They play a key role in morphogenesis, patterning, and lineage decision during central and peripheral nervous system development (3). Wnt ligands control gene expression (4), and their dysregulation has been implicated in cancer and major neuropathologies (5–9).The sustained expression of Wnt ligands and Wnt signaling components in the mature mammalian CNS and their involvement in neuropathologies suggest that these signaling cascades might also play a part in synaptic maintenance and function beyond embryonic development (10, 11). However, because of the pleiotropy and complexity of Wnt signaling, it has been difficult to dissect the components of Wnt signaling present in mature neurons and their role, if any, in the regulation of established synaptic connections and synaptic transmission.Although most excitatory glutamatergic neurotransmission in the brain is mediated by AMPA-type glutamate receptors [i.e., AMPA receptors (AMPARs)], unique properties allow the NMDA-type glutamate receptors [i.e., NMDA receptors (NMDARs)] to play a critical role in synaptic plasticity, learning and memory, and the establishment and maturation of functional neural circuits (12–14). Despite their importance, it is not known what controls the number and subunit composition of synaptic NMDARs. Dysfunction of NMDARs has been implicated in numerous diseases, including Huntington disease, Parkinson disease, depression, bipolar disorder, and schizophrenia (14, 15), in which a deficit in NMDAR mediated neurotransmission may be central (16). Interestingly, NMDAR-mediated currents can be acutely and specifically up-regulated by Wnt5a, a noncanonical Wnt ligand (17), but little is known regarding the signaling pathway and mechanisms involved in such regulation.The receptor tyrosine kinase-like orphan receptor 2 (RoR2) is part of a conserved family of tyrosine kinase-like receptors that have been proposed to serve as a receptor for noncanonical Wnt ligands, participating in developmental processes like cell movement and cell polarity (18, 19). Although RoR2 protein has been detected in mammalian neurons (20), its function and signaling pathways are not known. Here we show that RoR2 acts as a receptor for noncanonical Wnt ligands capable of regulating synaptic NMDARs. In hippocampal neurons, activation of RoR2 by noncanonical Wnt ligand Wnt5a activates PKC and JNK, two kinases involved in the regulation of NMDAR currents. In addition, we show that signaling through RoR2 is necessary for the maintenance of basal NMDAR-mediated synaptic transmission and the acute regulation of NMDAR synaptic responses by Wnt5a.Identification of RoR2 as a Wnt receptor that regulates synaptic NMDARs provides a mechanism for Wnt signaling to control synaptic transmission and synaptic plasticity acutely, and is a critical first step toward understanding the role played by Wnt signaling in the regulation of glutamatergic synaptic function under normal or pathological conditions.     

Serine/threonine kinase PKR: a sentinel kinase that discriminates a signaling pathway mediated by TLR4 from those mediated by TLR3 and TLR9

Cells of the innate immune system discriminate between "noninfectious self" and "infectious nonself" via pattern recognition receptors known as Toll-like receptors (TLRs). Though TLRs and the related interleukin 1 receptors share considerable homology in their cytoplasmic domains and adaptor molecules, signaling cascades may substantially differ from one another depending on the adaptor proteins recruited. Here we show that ectopic overexpression of catalytically inactive dominant-negative PKR expression system suppressed NF- kappa B activation mediated by TLR3, TLR9, TNF receptor 1 and 2 (TNF-R 1/2), but not by TLR4. Physiological relevance of the observations described here are discussed.     

Long‐term safety and efficacy of tofogliflozin as add‐on to insulin in patients with type 2 diabetes: Results from a 52‐week,multicentre, randomized,double‐blind,open‐label extension,Phase 4 study in Japan (J‐STEP/INS)

Aims

To evaluate the long‐term safety and efficacy of tofogliflozin as an add‐on treatment to insulin over 52 weeks.

Materials and methods

This 52‐week, multicentre, Phase 4 study consisted of a 16‐week, randomized, double‐blind, placebo‐controlled phase and a 36‐week open label extension phase (NCT02201004). Japanese patients with type 2 diabetes mellitus, aged 20 to 75 years, with suboptimal glycaemic control (7.5%‐10.5%) receiving insulin monotherapy (basal‐bolus, bolus, premix [low and high] and basal) or receiving combination therapy with basal insulin and dipeptidyl peptidase‐4 inhibitor were eligible for participation. Patients who received tofogliflozin throughout the study (52 weeks) were referred to as the ‘tofo‐tofo group’ and patients who received placebo and tofogliflozin (36 weeks) were referred to as the ‘pla‐tofo group’.

Results

A total of 210 patients received treatment per randomization. Hypoglycaemia was the most common treatment‐emergent adverse event (AE) (42.9% in the tofo‐tofo group and 29.4% in the pla‐tofo group). Patients reported genital infection, urinary tract infection, excessive urination and AEs related to volume depletion (2.1%, 2.1%, 7.1% and 10.0% of patients in the tofo‐tofo group, and 0%, 1.5%, 2.9% and 7.4% of patients in the pla‐tofo group, respectively). Mean HbA1c and body weight at baseline (mean changes ± standard error from baseline to Week 52) in the tofo‐tofo and pla‐tofo groups were 8.53% (?0.76% ± 0.077) and 8.40% (?0.73% ± 0.102); 68.84 kg (?1.52 kg ± 0.207) and 72.24 kg (?2.13 kg ± 0.313), respectively.

Conclusions

This study demonstrates the safety and efficacy of tofogliflozin as add‐on to insulin therapy in type 2 diabetes mellitus patients, offering a new therapeutic solution to diabetes management.