Oxidized phosphatidylcholines induce multiple functional defects in airway epithelial cells.

Oxidative stress is a hallmark of numerous airway diseases, contributing to extensive cell and tissue damage. Cell membranes and the airway mucosal lining are rich in phospholipids that are particularly susceptible to oxidative attack, producing bioactive molecules including oxidized phosphatidylcholines (OxPCs). With the recent discovery of elevated OxPCs in patients with asthma after allergen challenge, we hypothesized that OxPCs directly contribute to disease by inducing airway epithelial cell dysfunction. We found that OxPCs induced concentration-dependent cell stress and loss of viability in BEAS-2B and Calu-3 cell lines and primary human epithelial cells. These responses corresponded with significant epithelial barrier dysfunction, which was further compounded when combining OxPCs with an epithelial wound. OxPCs inhibited DNA synthesis and migration required to reestablish barrier function, but cells recovered if OxPCs were washed off soon after treatment. OxPCs induced generation of reactive oxygen species, lipid peroxidation, and mitochondrial dysfunction, raising the possibility that OxPCs cause pathological lipid metabolism in a self-propagating cycle. The oxidative stress induced by OxPCs could not be abrogated by putative OxPC receptor blockers, but partial recovery of barrier function, proliferation, and lipid peroxidation could be achieved with the antioxidant N-acetyl cysteine. In summary, we have identified OxPCs as a group of bioactive molecules that significantly impair multiple facets of epithelial cell function, consistent with pathological features of asthma. Further characterization of the mechanisms by which OxPCs affect epithelial cells could yield new insights into how oxidative stress contributes to the pathogenesis of airway disease.

Inflammatory cytokines IL-32 and IL-17 have common signaling intermediates despite differential dependence on TNF-receptor 1.

Cytokines IL-32 and IL-17 are emerging as critical players in the pathophysiology of immune-mediated chronic inflammatory diseases. It has been speculated that the molecular mechanisms governing IL-32- and IL-17-mediated cellular responses are differentially dependent on the TNF pathway. In this study, kinome analysis demonstrated that following stimulation with cytokine IL-32, but not IL-17, there was increased phosphorylation of a peptide target corresponding to TNF-R1. Consistent with this observation, blocking TNF-R1 resulted in a suppression of IL-32-induced downstream responses, indicating that IL-32-mediated activity may be dependent on TNF-R1. In contrast, blocking TNF-R1 did not affect IL-17-induced downstream responses. Kinome analysis also implicated p300 (transcriptional coactivator) and death-associated protein kinase-1 (DAPK-1) as signaling intermediates for both IL-32 and IL-17. Phosphorylation of p300 and DAPK-1 upon stimulation with either IL-32 or IL-17 was confirmed by immunoblots. The presence of common targets was supported by results demonstrating similar downstream responses induced in the presence of IL-32 and IL-17, such as transcriptional responses and the direct activation of NF-κB. Furthermore, knockdown of p300 and DAPK-1 altered downstream responses induced by IL-32 and IL-17, and impacted certain cellular responses induced by TNF-α and IL-1ß. We hypothesize that p300 and DAPK-1 represent nodes where the inflammatory networks of IL-32 and IL-17 overlap, and that these proteins would affect both TNF-R1-dependent and -independent pathways. Therefore, p300 and DAPK-1 are viable potential therapeutic targets for chronic inflammatory diseases.

Magnesium sulfate treatment for juvenile ferrets following induction of hydrocephalus with kaolin.

BACKGROUND: Previous work with 3-week hydrocephalic rats showed that white matter damage could be reduced by the calcium channel antagonist magnesium sulfate (MgSO4). We hypothesized that MgSO4 therapy would improve outcomes in ferrets with hydrocephalus induced with kaolin at 15 days. METHODS: MRI was performed at 29 days to assess ventricle size and stratify ferrets to treatment conditions. Beginning at 31 days age, they were treated daily for 14 days with MgSO4 (9 mM/kg/day) or sham saline therapy, and then imaged again before sacrifice. Behavior was examined thrice weekly. Histological and biochemical ELISA and myelin enzyme activity assays were performed at 46 days age. RESULTS: Hydrocephalic ferrets exhibited some differences in weight and behavior between treatment groups. Those receiving MgSO4 weighed less, were more lethargic, and displayed reduced activity compared to those receiving saline injections. Hydrocephalic ferrets developed ventriculomegaly, which was not modified by MgSO4 treatment. Histological examination showed destruction of periventricular white matter. Glial fibrillary acidic protein content, myelin basic protein content, and myelin enzyme activity did not differ significantly between treatment groups. CONCLUSION: The hydrocephalus-associated disturbances in juvenile ferret brains are not ameliorated by MgSO4 treatment, and lethargy is a significant side effect.

Vitamin D, serum 25(OH)D, LL-37 and polymorphisms in a Canadian First Nation population with endemic tuberculosis.

BACKGROUND: Canadian First Nation populations have experienced endemic and epidemic tuberculosis (TB) for decades. Vitamin D-mediated induction of the host defence peptide LL-37 is known to enhance control of pathogens such as Mycobacterium tuberculosis. OBJECTIVE: Evaluate associations between serum levels of 25-hydroxy vitamin D (25(OH)D) and LL-37, in adult Dene First Nation participants (N = 34) and assess correlations with single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) and vitamin D binding protein (VDBP). DESIGN: Venous blood was collected from all participants at baseline (winter and summer) and in conjunction with taking vitamin D supplements (1,000 IU/day) (winter and summer). Samples were analysed using ELISA for concentrations of vitamin D and LL-37, and SNPs in the VDR and VDBP regions were genotyped. RESULTS: Circulating levels of 25(OH)D were not altered by vitamin D supplementation, but LL-37 levels were significantly decreased. VDBP and VDR SNPs did not correlate with serum concentrations of 25(OH)D, but LL-37 levels significantly decreased in individuals with VDBP D432E T/G and T/T, and with VDR SNP Bsm1 T/T genotypes. CONCLUSIONS: Our findings suggest that vitamin D supplementation may not be beneficial as an intervention to boost innate immune resistance to M. tuberculosis in the Dene population.

Oral antioxidant therapy for juvenile rats with kaolin-induced hydrocephalus.

BACKGROUND: Oxidative and nitrosylative changes have been shown to occur in conjunction with the hypoxic changes and cellular/axonal damage in hydrocephalic rodent brains. We hypothesized that antioxidant therapy would improve behavioral, neurophysiological, and/or neurobiochemical outcomes in juvenile rats following induction of hydrocephalus. METHODS: Three-week old rats received an injection of kaolin (aluminum silicate) into the cisterna magna. Magnetic resonance (MR) imaging was performed two weeks later to assess ventricle size and stratify rats to four treatment conditions. Rats were treated for two weeks daily with sham therapy of either oral canola oil or dextrose or experimental therapy of a low or high dose of an antioxidant mixture containing α-tocopherol, L-ascorbic acid, coenzyme Q10 (CoQ10), reduced glutathione, and reduced lipoic acid. Behavior was examined thrice weekly. RESULTS: All hydrocephalic groups lagged in weight gain in comparison to non-hydrocephalic controls, all developed significant ventriculomegaly, and all exhibited white matter destruction. Canola oil with or without the antioxidant mixture normalized antioxidant capacity in brain tissue, and the dextrose-treated rats had the greatest ventricular enlargement during the treatment period. However, there were no significant differences between the four treatment groups of hydrocephalic rats for the various behavioral tasks. Glial fibrillary acidic protein and myelin basic protein quantitation showed no differences between the treatment groups or with control rats. There was increased lipid peroxidation in the hydrocephalic rats compared to controls but no differences between treatment groups. CONCLUSION: The antioxidant cocktail showed no therapeutic benefits for juvenile rats with kaolin-induced hydrocephalus although canola oil might have mild benefit.

Effect of vitamin D supplementation on Mycobacterium tuberculosis-induced innate immune responses in a Canadian Dené First Nations cohort.

Canadian First Nations (FN) population experiences a high burden of tuberculosis. Vitamin D is known to enhance the expression of innate immune effectors, including cathelicidin LL-37, for protection against infections. In this study we performed longitudinal analyses to investigate the impact of vitamin D supplementation on macrophage responses to Mycobacterium tuberculosis (Mtb) lipoprotein (TLR2/1L), in Canadian Dené FN participants compared to Caucasian participants. Serum 25(OH)D and LL-37 levels were evaluated by ELISA. Transcriptional responses and protein expression of TLR2/1L-induced LL-37 and other innate immune cytokines were monitored in monocyte-derived macrophages (MDMs) before and after 8 months of vitamin D supplementation. In this study we showed that serum levels of LL-37 decreased after vitamin D supplementation in both Dené and Caucasian participants. There was no difference in TLR2/1L-induced LL-37 expression in MDMs in the two groups, either pre- or post-vitamin D supplementation. However, vitamin D supplementation markedly enhanced TLR2/1L-induced responses in MDMs e.g. IL-6, IL-12 and IL-23 among Caucasians but not in the Dené participants. In contrast, after vitamin D supplementation TLR2/1L-induced responses e.g. IL-1ß, IL-8 and IL-12 were significantly reduced in the Dené MDMs. These results indicate that vitamin D supplementation enhanced TLR2/1L-induced innate immune macrophage responses in the Caucasian but not in the Dené participants. We hypothesize that cytokines may be differentially regulated in Canadian FN compared to Caucasians, in particular those that influence Th-1 and Th-17 responses required for the control of Mtb.

Vitamin D in a northern Canadian first nation population: dietary intake, serum concentrations and functional gene polymorphisms.

The wide spectrum of vitamin D activity has focused attention on its potential role in the elevated burden of disease in a northern Canadian First Nations (Dené) cohort. Vitamin D insufficiency, and gene polymorphisms in the vitamin D receptor (VDR) and vitamin D binding protein (VDBP) have been implicated in susceptibility to infectious and chronic diseases. The objectives of this study were to determine the contribution of vitamin D from food, and measure the serum concentrations of 25-hydroxyvitamin D(3) (25-OHD(3)) and VDBP in Dené participants. Single nucleotide polymorphisms (SNPs) associated with the dysregulation of the innate immune response were typed and counted. Potential correlations between the SNPs and serum concentrations of 25-OHD(3) and VDBP were evaluated. Venous blood was collected in summer and winter over a one-year period and analyzed for 25-OHD(3) and VDBP concentrations (N = 46). A questionnaire was administered to determine the amount of dietary vitamin D consumed. Sixty-one percent and 30% of the participants had 25-OHD(3) serum concentrations <75 nmol/L in the winter and summer respectively. Mean vitamin D binding protein concentrations were within the normal range in the winter but below normal in the summer. VDBP and VDR gene polymorphisms affect the bioavailability and regulation of 25-OHD(3). The Dené had a high frequency of the VDBP D432E-G allele (71%) and the Gc1 genotype (90%), associated with high concentrations of VDBP and a high binding affinity to 25-OHD(3). The Dené had a high frequency of VDR Fok1-f allele (82%), which has been associated with a down-regulated Th1 immune response. VDBP and VDR polymorphisms, and low winter 25-OHD(3) serum concentrations may be risk factors for infectious diseases and chronic conditions related to the dysregulation of the vitamin D pathway.

Modulation of interleukin-1ß-induced inflammatory responses by a synthetic cationic innate defence regulator peptide, IDR-1002, in synovial fibroblasts.

INTRODUCTION: Innate defence regulator (IDR) peptides are synthetic cationic peptides, variants of naturally occurring innate immune effector molecules known as host defence peptides. IDR peptides were recently demonstrated to limit infection-associated inflammation selectively without compromising host innate immune functions. This study examined the impact of a 12-amino acid IDR peptide, IDR-1002, in pro-inflammatory cytokine interleukin (IL)-1ß-induced responses in synovial fibroblasts, a critical cell type in the pathogenesis of inflammatory arthritis. METHODS: Human fibroblast-like synoviocytes (FLS) were stimulated with IL-1ß in the presence and absence of IDR-1002. Production of enzyme matrix metalloproteinase-3 (MMP-3) and IL-1-receptor antagonist (IL-1RA) was monitored by enzyme-linked immunosorbent assay (ELISA), and various chemokines were evaluated by using multiplex cytometric bead array. Transcriptional responses were analyzed by quantitative real-time PCR. The impact on IL-1ß-induced proteome was investigated by quantitative proteomics by using isobaric tags. IL-1ß-induced pathways altered by IDR-1002 implicated by the proteomics analyses were further investigated by using various immunochemical assays. Cellular uptake of the peptide was monitored by using a biotinylated IDR-1002 peptide followed by microscopy probing with streptavidin-Alexa Fluor. RESULTS: This study demonstrated that IDR-1002 suppressed the production of IL-1ß-induced MMP-3 and monocyte chemotactic protein-1 (MCP-1); in contrast, IDR-1002 enhanced the production of IL-1RA, without neutralizing all chemokine responses. IDR-1002 altered the IL-1ß-induced proteome primarily by altering the expression of members of nuclear factor kappa-B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways. The proteomics data also suggested that IDR-1002 was altering the transcription factor HNF-4α-mediated responses, known to be critical in metabolic regulation. With various immunochemical assays, it was further demonstrated that IL-1ß-induced NF-κB, JNK, and p38 mitogen-activated protein kinase (MAPK) activations were significantly suppressed by IDR-1002. CONCLUSIONS: This study demonstrates the ability of an innate immune-modulatory IDR-peptide to influence the IL-1ß-induced regulatory pathways and selectively to suppress inflammatory responses in synovial fibroblasts. The results of this study provide a rationale for examining the use of IDR-peptides as potential therapeutic candidates for chronic inflammatory diseases such as inflammatory arthritis.