Catherine M. Greene

Signal transduction pathways activated by the IL-1 receptor family: ancient signaling machinery in mammals, insects, and plants.

Interleukin‐1 (IL‐1) is a central regulator of the immune and inflammatory responses. Recently, significant advances have been made in the area of IL‐1 receptors and IL‐1 signal transduction. A family of proteins has been described that share significant homology in their signaling domains with the Type I IL‐1 receptor (IL‐1RI). These include the IL‐1 receptor accessory protein (IL‐1AcP), which does not bind IL‐1 but is essential for IL‐1 signaling; a Drosophila protein Toll; a number of human Toll‐like receptors (hTLRs); the putative IL‐18/IL‐1‐γ receptor IL‐1Rrp (IL‐1 receptor‐related protein); and a number of plant proteins. All appear to be involved in host responses to injury and infection. These homologies also extend to novel signaling proteins implicated in IL‐1 action. Two IL‐1 receptor‐associated kinases, IRAK‐1 and IRAK‐2, which have homologs in Drosophila (Pelle) and plants (Pto), have been implicated in the activation of the transcription factor, nuclear factor κB (NF‐κB). IRAK‐1 has also been implicated in AP1 induction, Jun amino‐terminal kinase (JNK) activation, and IL‐2 induction. It recruits the adapter protein TRAF6 to the IL‐1 receptor complex via an interaction with IL‐1AcP. TRAF6 then relays the signal via NF‐κB‐inducing kinase (NIK) to two I‐κB kinases (IKK‐1 and ‐2), leading to NF‐κB activation. Progress has also been made on other IL‐1‐responsive kinases, including JNK and p38 MAP kinase, with the latter having a role in multiple responses to IL‐1. The remarkable conservation between diverse species indicates that the IL‐1 system represents an ancient signaling machine critical for responses to environmental stresses and attack by pathogens. J. Leukoc. Biol. 63: 650–657; 1998.

Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes

Staphylococcus aureus 8325‐4 has the potential to express two distinct cell wall‐associated fibronectin‐binding proteins called FnBPA and FnBPB. In order to test if both proteins are expressed in S. aureus and if both are required for promoting bacterial adhesion to fibronectin‐coated surfaces, insertion mutations were isolated in each gene. A DNA fragment encoding tetracycline resistance was inserted into fnbA and a fragment encoding erythromycin resistance was inserted into fnbB. A double fnbA fnbB mutant was also constructed. The fnbA and fnbB single mutants showed no significant reduction in their adhesion to polymethylmethacrylate coverslips that had been coated in vitro with fibronectin. However, the double mutant was completely defective in adhesion. Monospecific antibodies directed against the non‐conserved N‐terminal regions of both proteins confirmed the lack of expression of FnBPs in the mutant strains. Wild‐type fnbA and fnbB genes cloned seperately on a multicopy plasmid were each able to restore fully the adhesion‐defective phenotype of the 8325‐4 fnbA fnbB mutant. This demonstrates that both fnb genes are expressed in S. aureus and that both contribute to the ability of strain 8325‐4 to adhere to fibronectin‐coated surfaces. The double mutant was also defective in adhesion to coverslips that had been removed from tissue cages implanted subcutaneously in guinea‐pigs, which suggests that fibronectin is important in promoting attachment of S. aureus to biomaterial in vivo.

Neutrophil elastase up-regulates interleukin-8 via toll-like receptor 4.

Cystic fibrosis is characterised in the lungs by high levels of neutrophil elastase (NE). NE induces interleukin‐8 (IL‐8) expression via an IL‐1 receptor‐associated kinase signalling pathway. Here, we show that these events involve the cell surface membrane bound toll‐like receptor 4 (TLR4). We demonstrate that human embryonic kidney (HEK)293 cells transfected with a TLR4 cDNA (HEK‐TLR4) express TLR4 mRNA and protein and induce IL‐8 promoter activity in response to NE. Treatment of both HEK‐TLR4 and human bronchial epithelial cells with NE decreases TLR4 protein expression. Furthermore, a TLR4 neutralising antibody abrogates NE‐induced IL‐8 production, and induces tolerance to a secondary lipopolysaccharide stimulus. These data implicate TLR4 in NE induced IL‐8 expression in bronchial epithelium.

TLR-Induced Inflammation in Cystic Fibrosis and Non-Cystic Fibrosis Airway Epithelial Cells

Cystic fibrosis (CF) is a genetic disease characterized by severe neutrophil-dominated airway inflammation. An important cause of inflammation in CF is Pseudomonas aeruginosa infection. We have evaluated the importance of a number of P. aeruginosa components, namely lipopeptides, LPS, and unmethylated CpG DNA, as proinflammatory stimuli in CF by characterizing the expression and functional activity of their cognate receptors, TLR2/6 or TLR2/1, TLR4, and TLR9, respectively, in a human tracheal epithelial line, CFTE29o−, which is homozygous for the ΔF508 CF transmembrane conductance regulator mutation. We also characterized TLR expression and function in a non-CF airway epithelial cell line 16HBE14o−. Using RT-PCR, we demonstrated TLR mRNA expression. TLR cell surface expression was assessed by fluorescence microscopy. Lipopeptides, LPS, and unmethylated CpG DNA induced IL-8 and IL-6 protein production in a time- and dose-dependent manner. The CF and non-CF cell lines were largely similar in their TLR expression and relative TLR responses. ICAM-1 expression was also up-regulated in CFTE29o− cells following stimulation with each agonist. CF bronchoalveolar lavage fluid, which contains LPS, bacterial DNA, and neutrophil elastase (a neutrophil-derived protease that can activate TLR4), up-regulated an NF-κB-linked reporter gene and increased IL-8 protein production in CFTE29o− cells. This effect was abrogated by expression of dominant-negative versions of MyD88 or Mal, key signal transducers for TLRs, thereby implicating them as potential anti-inflammatory agents for CF.

Association of IL-10 polymorphism with severity of illness in community acquired pneumonia.

Background: The influence of genetic polymorphisms of interleukin (IL)-10, tumour necrosis factor (TNF)-α, and IL-6 gene promoters on severity of systemic inflammatory response syndrome (SIRS) associated with community acquired pneumonia (CAP) was studied. Methods: Using PCR-RFLP analysis we analysed a −1082G/A single nucleotide polymorphism (SNP) of the anti-inflammatory IL-10 gene, a −308G/A SNP of the pro-inflammatory TNF-α gene and a −174G/C SNP of the IL-6 gene. Illness severity was stratified according to SIRS score, calculated by presence of up to four physiological indices: temperature, white blood cell count, heart rate and respiratory rate (non-SIRS, SIRS 2, SIRS 3, and SIRS 4). Results: A statistically significant stepwise increase in frequency of the IL-10 G allele, associated with higher expression of the gene, was observed in patients with increasing severity of illness from non-SIRS (n=19) to SIRS 2 (n=17), SIRS 3 (n=33) and SIRS 4 (n=24). This was primarily due to a higher frequency of the GG genotype with increasing severity from non-SIRS through to SIRS 4. IL-10 G allele frequency was also increased in patients who died as a result of CAP (n=11) compared with CAP survivors (n=82) (p=0.01). No association was seen between the TNF-α −308G/A and IL-6 −174G/C SNPs and disease. Additionally, no interaction between all three SNP genotypes and disease severity was observed. Conclusions: A polymorphism affecting IL-10 expression may influence the severity of illness in patients with CAP.

Inactivation of Human β-Defensins 2 and 3 by Elastolytic Cathepsins

β-Defensins are antimicrobial peptides that contribute to the innate immune responses of eukaryotes. At least three defensins, human β-defensins 1, 2, and 3 (HBD-1, -2, and -3), are produced by epithelial cells lining the respiratory tract and are active toward Gram-positive (HBD-3) and Gram-negative (HBD-1, -2, and -3) bacteria. It has been postulated that the antimicrobial activity of defensins is compromised by changes in airway surface liquid composition in lungs of patients with cystic fibrosis (CF), therefore contributing to the bacterial colonization of the lung by Pseudomonas and other bacteria in CF. In this report we demonstrate that HBD-2 and HBD-3 are susceptible to degradation and inactivation by the cysteine proteases cathepsins B, L, and S. In addition, we show that all three cathepsins are present and active in CF bronchoalveolar lavage. Incubation of HBD-2 and -3 with CF bronchoalveolar lavage leads to their degradation, which can be completely (HBD-2) or partially (HBD-3) inhibited by a cathepsin inhibitor. These results suggest that β-defensins are susceptible to degradation and inactivation by host proteases, which may be important in the regulation of β-defensin activity. In chronic lung diseases associated with infection, overexpression of cathepsins may lead to increased degradation of HBD-2 and -3, thereby favoring bacterial infection and colonization.

Activation of Endoplasmic Reticulum-Specific Stress Responses Associated with the Conformational Disease Z α1-Antitrypsin Deficiency

Conformational diseases are a class of disorders associated with aberrant protein accumulation in tissues and cellular compartments. Z α1-antitrypsin (A1AT) deficiency is a genetic disease associated with accumulation of misfolded A1AT in the endoplasmic reticulum (ER) of hepatocytes. We sought to identify intracellular events involved in the molecular pathogenesis of Z A1AT-induced liver disease using an in vitro model system of Z A1AT ER accumulation. We investigated ER stress signals induced by Z A1AT and demonstrated that both the ER overload response and the unfolded protein response were activated by mutant Z A1AT, but not wild-type M A1AT. Interestingly, activation of the unfolded protein response pathway required an additional insult, whereas NF-κB activation, a hallmark of the ER overload response, was constitutive. These findings have important implications for the design of future therapeutics for Z A1AT liver disease and may also impact on drug design for other conformational diseases.

Interleukin-8 Up-regulation by Neutrophil Elastase Is Mediated by MyD88/IRAK/TRAF-6 in Human Bronchial Epithelium

Cystic fibrosis is characterized in the lungs by neutrophil-dominated inflammation mediated significantly by neutrophil elastase (NE). Previous work has shown that NE induces interleukin-8 (IL-8) gene expression and protein secretion in bronchial epithelial cells. We sought to determine the intracellular mechanisms by which NE up-regulates IL-8 in bronchial epithelial cells. The data show that stimulation of 16HBE14o− cells with NE induced IL-8 protein production and gene expression. Both responses were abrogated by actinomycin D, indicating that regulation is at the transcriptional level. Electrophoretic mobility shift assays demonstrated that nuclear factor κB (NFκB) was activated in 16HBE14o− cells stimulated with NE. Western blot analysis demonstrated that activation of NFκB by NE was preceded by phosphorylation and degradation of IκB proteins, principally IκBβ. In addition, we observed that interleukin-1 receptor-associated kinase (IRAK) was degraded in 16HBE14o− cells stimulated with NE. Quantification of IL-8 reporter gene activity by luminometry demonstrated that dominant negative MyD88 (MyD88Δ) or TRAF-6 (TRAF-6Δ) inhibited IL-8 reporter gene expression in response to NE. Furthermore, MyD88Δ inhibited NE-induced IRAK degradation. These results show that NE induces IL-8 gene up-regulation in bronchial epithelial cells through an IRAK signaling pathway involving both MyD88 and TRAF-6, resulting in degradation of IκBβ and nuclear translocation of NFκB. These findings may have implications for therapeutic treatments in the cystic fibrosis condition.

Innate immunity in cystic fibrosis lung disease

Chronic lung disease determines the morbidity and mortality of cystic fibrosis (CF) patients. The pulmonary immune response in CF is characterized by an early and non-resolving activation of the innate immune system, which is dysregulated at several levels. Here we provide a comprehensive overview of innate immunity in CF lung disease, involving (i) epithelial dysfunction, (ii) pathogen sensing, (iii) leukocyte recruitment, (iv) phagocyte impairment, (v) mechanisms linking innate and adaptive immunity and (iv) the potential clinical relevance. Dissecting the complex network of innate immune regulation and associated pro-inflammatory cascades in CF lung disease may pave the way for novel immune-targeted therapies in CF and other chronic infective lung diseases.

miR-126 Is Downregulated in Cystic Fibrosis Airway Epithelial Cells and Regulates TOM1 Expression

Cystic fibrosis (CF) is one of the most common lethal genetic diseases in which the role of microRNAs has yet to be explored. Predicted to be regulated by miR-126, TOM1 (target of Myb1) has been shown to interact with Toll-interacting protein, forming a complex to regulate endosomal trafficking of ubiquitinated proteins. TOM1 has also been proposed as a negative regulator of IL-1β and TNF-α–induced signaling pathways. MiR-126 is highly expressed in the lung, and we now show for the first time differential expression of miR-126 in CF versus non-CF airway epithelial cells both in vitro and in vivo. MiR-126 downregulation in CF bronchial epithelial cells correlated with a significant upregulation of TOM1 mRNA, both in vitro and in vivo when compared with their non-CF counterparts. Introduction of synthetic pre–miR-126 inhibited luciferase activity in a reporter system containing the full length 3′-untranslated region of TOM1 and resulted in decreased TOM1 protein production in CF bronchial epithelial cells. Following stimulation with LPS or IL-1β, overexpression of TOM1 was found to downregulate NF-κB luciferase activity. Conversely, TOM1 knockdown resulted in a significant increase in NF-κB regulated IL-8 secretion. These data show that miR-126 is differentially regulated in CF versus non-CF airway epithelial cells and that TOM1 is a miR-126 target that may have an important role in regulating innate immune responses in the CF lung. To our knowledge, this study is the first to report of a role for TOM1 in the TLR2/4 signaling pathways and the first to describe microRNA involvement in CF.