Sha-Mei Liao

IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway.

The transcription factors interferon regulatory factor 3 (IRF3) and NF-κB are required for the expression of many genes involved in the innate immune response. Viral infection, or the binding of double-stranded RNA to Toll-like receptor 3, results in the coordinate activation of IRF3 and NF-κB. Activation of IRF3 requires signal-dependent phosphorylation, but little is known about the signaling pathway or kinases involved. Here we report that the noncanonical IκB kinase homologs, IκB kinase-ε (IKKε) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-κB activation, are also essential components of the IRF3 signaling pathway. Thus, IKKε and TBK1 have a pivotal role in coordinating the activation of IRF3 and NF-κB in the innate immune response.

IKKε Is Part of a Novel PMA-Inducible IκB Kinase Complex

Here we report the identification of a novel PMA-inducible IkappaB kinase complex, distinct from the well-characterized high-molecular weight IkappaB kinase complex containing IKKalpha, IKKbeta, and IKKgamma. We have characterized one kinase from this complex, which we designate IKKepsilon. Although recombinant IKKepsilon directly phosphorylates only serine 36 of IKBalpha, the PMA-activated endogenous IKKepsilon complex phosphorylates both critical serine residues. Remarkably, this activity is due to the presence of a distinct kinase in this complex. A dominant-negative mutant of IKKepsilon blocks induction of NF-kappaB by both PMA and activation of the T cell receptor but has no effect on the activation of NF-KB by TNFalpha or IL-1. These observations indicate that the activation of NF-kappaB requires multiple distinct IkappaB kinase complexes, which respond to both overlapping and discrete signaling pathways.

Validation of the Anti-Inflammatory Properties of Small-Molecule IκB Kinase (IKK)-2 Inhibitors by Comparison with Adenoviral-Mediated Delivery of Dominant-Negative IKK1 and IKK2 in Human Airways Smooth Muscle

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by chronic airway inflammation. However, because patients with COPD and certain patients with asthma show little or no therapeutic benefit from existing corticosteroid therapies, there is an urgent need for novel anti-inflammatory strategies. The transcription factor nuclear factor-κB (NF-κB) is central to inflammation and is necessary for the expression of numerous inflammatory genes. Proinflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor (TNF)-α, activate the IκB kinase complex (IKK) to promote the degradation of inhibitory IκB proteins and activate NF-κB. This pathway and, in particular, the main IκB kinase, IKK2, are now considered prime targets for novel anti-inflammatory drugs. Therefore, we have used adenoviral overexpression to demonstrate NF-κB and IKK2 dependence of key inflammatory genes, including intercellular adhesion molecule (ICAM)-1, cyclooxygenase-2, IL-6, IL-8, granulocyte macrophage-colony-stimulating factor (GM-CSF), regulated on activation normal T cell expressed and secreted (RANTES), monocyte chemotactic protein-1 (MCP-1), growth-regulated oncogene-α (GROα), neutrophil-activating protein-2 (NAP-2), and epithelial neutrophil activating peptide 78 (ENA-78) in primary human airways smooth muscle cells. Because this cell type is central to the pathogenesis of airway inflammatory diseases, these data predict a beneficial effect of IKK2 inhibition. These validated outputs were therefore used to evaluate the novel IKK inhibitors N-(6-chloro-9H-β-carbolin-8-yl) nicotinamide (PS-1145) and N-(6-chloro-7-methoxy-9H-β-carbolin-8-yl)-2-methyl-nicotinamide (ML120B) on IL-1β and TNFα-induced expression, and this was compared with the corticosteroid dexamethasone. As observed above, ICAM-1, IL-6, IL-8, GM-CSF, RANTES, MCP-1, GROα, NAP-2, and ENA-78 expression was reduced by the IKK inhibitors. Furthermore, this inhibition was either as effective, or for ICAM-1, MCP-1, GROα, and NAP-2, more effective, than a maximally effective concentration of dexamethasone. We therefore suggest that IKK inhibitors may be of considerable benefit in inflammatory airways diseases, particularly in COPD or severe asthma, in which corticosteroids are ineffective.

Small-molecule factor D inhibitors targeting the alternative complement pathway

Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.

Lack of Involvement of CEP Adducts in TLR Activation and in Angiogenesis

Proteins that are post-translationally adducted with 2-(ω-carboxyethyl)pyrrole (CEP) have been proposed to play a pathogenic role in age-related macular degeneration, by inducing angiogenesis in a Toll Like Receptor 2 (TLR2)-dependent manner. We have investigated the involvement of CEP adducts in angiogenesis and TLR activation, to assess the therapeutic potential of inhibiting CEP adducts and TLR2 for ocular angiogenesis. As tool reagents, several CEP-adducted proteins and peptides were synthetically generated by published methodology and adduction was confirmed by NMR and LC-MS/MS analyses. Structural studies showed significant changes in secondary structure in CEP-adducted proteins but not the untreated proteins. Similar structural changes were also observed in the treated unadducted proteins, which were treated by the same adduction method except for one critical step required to form the CEP group. Thus some structural changes were unrelated to CEP groups and were artificially induced by the synthesis method. In biological studies, the CEP-adducted proteins and peptides failed to activate TLR2 in cell-based assays and in an in vivo TLR2-mediated retinal leukocyte infiltration model. Neither CEP adducts nor TLR agonists were able to induce angiogenesis in a tube formation assay. In vivo, treatment of animals with CEP-adducted protein had no effect on laser-induced choroidal neovascularization. Furthermore, in vivo inactivation of TLR2 by deficiency in Myeloid Differentiation factor 88 (Myd88) had no effect on abrasion-induced corneal neovascularization. Thus the CEP-TLR2 axis, which is implicated in other wound angiogenesis models, does not appear to play a pathological role in a corneal wound angiogenesis model. Collectively, our data do not support the mechanism of action of CEP adducts in TLR2-mediated angiogenesis proposed by others.

Discovery of Highly Potent and Selective Small-Molecule Reversible Factor D Inhibitors Demonstrating Alternative Complement Pathway Inhibition in Vivo

The highly specific S1 serine protease factor D (FD) plays a central role in the amplification of the complement alternative pathway (AP) of the innate immune system. Genetic associations in humans have implicated AP activation in age-related macular degeneration (AMD), and AP dysfunction predisposes individuals to disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). The combination of structure-based hit identification and subsequent optimization of the center (S)-proline-based lead 7 has led to the discovery of noncovalent reversible and selective human factor D (FD) inhibitors with drug-like properties. The orally bioavailable compound 2 exerted excellent potency in 50% human whole blood in vitro and blocked AP activity ex vivo after oral administration to monkeys as demonstrated by inhibition of membrane attack complex (MAC) formation. Inhibitor 2 demonstrated sustained oral and ocular efficacy in a model of lipopolysaccharide (LPS)-induced systemic AP activation in mice expressing human FD.

Amount of Mononuclear Phagocyte Infiltrate Does Not Predict Area of Experimental Choroidal Neovascularization (CNV)

Abstract Purpose: Mononuclear phagocytes (MNPs) are present in neovascular age-related macular degeneration (nv AMD) which is also called choroidal neovascularization (CNV). The number and phenotype of the MNPs depend upon the local environment in the CNV and effect of nv AMD therapy. We investigated ocular cell infiltration and conditions that modulate angiogenesis in a laser-induced mouse CNV model. Methods: We developed assays to quantify MNPs in our established mouse CNV model. One MNP assay quantified the number of subretinal cells peripheral to the CNV lesions. A second assay semiquantitatively assesses the number of MNPs localized to the CNV lesion. We used these assays to measure the effect of toll-like receptor-2 (TLR-2) activation, anti-vascular endothelial growth factor (VEGF) therapy, and chemokine (C-C motif) ligand 2 (Ccl2) genetic deletion on MNP infiltration after laser injury. Results: Laser injury induced blood vessel growth and infiltration of MNPs. Systemic administration of a TLR-2 activating peptide increased laser-induced CNV area, MNP cell numbers, and MNP density over the CNV lesions. Systemic administration of a VEGF antibody reduced CNV area, while Ccl2 genetic deletion increased CNV area. Despite the change in amount of angiogenesis, MNP infiltration was, surprisingly, unchanged in these 2 conditions. Conclusions: MNP quantification provides biological insights for candidate AMD therapies. The number of infiltrating MNP cells does not correlate with the amount of laser-induced CNV area.

Induction of Ocular Complement Activation by Inflammatory Stimuli and Intraocular Inhibition of Complement Factor D in Animal Models

Purpose Genome-wide association studies suggest a role for the complement system in age-related macular degeneration (AMD). We characterized ocular complement activation and evaluated a complement factor D (FD) neutralizing antibody. Methods Mice were treated with toll-like receptor (TLR) ligands, intravitreal injection (IVT), or corneal debridement. Levels of complement proteins and mRNA were measured. A FD neutralizing antibody was administered IVT into eyes of rabbits that were challenged with LPS (lipopolysaccharide) administered intravenously. Results Levels of C3 and factor B (FB) mRNA and protein in the eye were increased following intraperitoneal injection of TLR4 ligand LPS. Increased levels of C3 and FB breakdown products were observed in both eye tissues and plasma. Complement activation products were markedly reduced in C3-/- and Cfb-/- mice challenged with LPS. Ocular complement levels were also elevated in mice treated systemically with TLR2 and -3 ligands, injured by IVT injection or corneal debridement, or even in normal aging. IVT administration of a complement FD neutralizing antibody in rabbits inhibited LPS-induced complement activation in the posterior segment of the eye, but not in the anterior segment of the eye or in plasma. Conclusions Systemic TLR stimulation and eye tissue injury induced time-dependent alternative complement pathway activation in the eye. Ocular complement levels were also gradually elevated during aging. An anti-FD antibody IVT potently inhibited LPS-induced complement activation in the posterior segment of the eye. This study provides insights into the dynamic profile of ocular complement activation, which is valuable for complement research in eye diseases and for developing complement therapeutics for AMD.