Proteomic analysis of a noninvasive human model of acute inflammation and its resolution:the twenty-one day gingivitis model

The 21-day experimental gingivitis model, an established noninvasive model of inflammation in response to increasing bacterial accumulation in humans, is designed to enable the study of both the induction and resolution of inflammation. Here, we have analyzed gingival crevicular fluid, an oral fluid comprising a serum transudate and tissue exudates, by LC-MS/MS using Fourier transform ion cyclotron resonance mass spectrometry and iTRAQ isobaric mass tags, to establish meta-proteomic profiles of inflammation-induced changes in proteins in healthy young volunteers. Across the course of experimentally induced gingivitis, we identified 16 bacterial and 186 human proteins. Although abundances of the bacterial proteins identified did not vary temporally, Fusobacterium outer membrane proteins were detected. Fusobacterium species have previously been associated with periodontal health or disease. The human proteins identified spanned a wide range of compartments (both extracellular and intracellular) and functions, including serum proteins, proteins displaying antibacterial properties, and proteins with functions associated with cellular transcription, DNA binding, the cytoskeleton, cell adhesion, and cilia. PolySNAP3 clustering software was used in a multilayered analytical approach. Clusters of proteins that associated with changes to the clinical parameters included neuronal and synapse associated proteins.

Mediators of monocyte activity in inflammation

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. CRP is currently one of the best markers of inflammatory disease and disease activity. One of the keys cells involved in inflammation within chronic inflammatory diseases is the monocyte. Monocytes are able to modulate inflammation through cytokine expression, cytosolic peroxide formation, adhesion molecule expression and subsequent adhesion/migration to sites of inflammation. CRP has been previously shown to bind directly to monocytes through Fc receptors. However this observation is not conclusive and requires further investigation. The effects of incubation of CRP with human primary and monocytic cell lines were examined using monocytic cytokine expression, adhesion molecule expression and adhesion to endothelial cells and intracellular peroxide formation, as end points. Monocytic intracellular signalling events were investigated after interaction of CRP with specific CRP receptors on monocytes. These initial signalling events were examined for their role in modulating monocytic adhesion molecule and cytokine expression. Monocyte recruitment and retention in the vasculature is also influenced by oxidative stress. Therefore the effect of 6 weeks of antioxidant intervention in vivo was examined on monocytic adhesion molecule expression, adhesion to endothelial cells ex vivo and on serum CRP concentrations, pre- and post- supplementation with the antioxidants vitamin C and vitaInin E. In summary, CRP is able to bind FcγRIIa. CRP binding FcγR initiates an intracellular signalling cascade that phosphorylates the non-receptor tyrosine kinase, Syk, associated with intracellular tyrosine activating motifs on the cytoplasmic tail of Fcγ receptors. CRP incubations increased phosphatidyl inositol turnover and Syk phosphorylation ultimately lead to Ca2+ mobilisation in monocytes. CRP mediated Syk phosphorylation in monocytes leads to an increase in CD 11b and IL-6 expression. CRP engagement with monocytes also leads to an increase in peroxide production, which can be inhibited in vitro using the antioxidants α-tocopherol and ascorbic acid. CRP mediated CD 11b expression is not redox regulated by CRP mediated changes in cytosolic peroxides. The FcyRIla polymorphism at codon 131 effects the phenotypic driven changes described in monocytes by CRP, where R/R allotypes have a greater increase in CD11b, in response to CRP, which may be involved in promoting the monocytic inflammatory response. CRP leads to an increase in the expression of pro-inflammatory cytokines, which alters the immune phenotype of circulating monocytes. Vitamin C supplementation reduced monocytic adhesion to endothelial cells, but had no effect on serum levels of CRP. Where long-term antioxidant intervention may provide benefit from the risk of developing vascular inflammatory disease, by reducing monocytic adhesion to the vasculature. In conclusion CRP appears to be much more than just a marker of ongoing inflammation or associated inflammatory disease and disease activity. This data suggests that at pathophysiological concentrations, CRP may be able to directly modulate inflammation through interacting with monocytes and thereby alter the inflammatory response associated with vascular inflammatory diseases.

Studies on bacterial lung infections in cystic fibrosis

The major cause of death in CF is a continuous inflammation of the lungs colonised with Pseudomonas aeruginosa and occasionally also with Burkholderia cepacia. A combination of serum IgG to LPS and serum PCT levels were found to be good markers for detection of early colonisation with P. aeruginosa. Colomycin sulphomethate (colistin E) is one of the antibiotics used to treat P. aeruginosa infections in CF. Electrophoretic methods were developed to monitor the rate of conversion of colomycin sulphomethate to the active form of the drug. Antimicrobial activity towards P. aeruginosa was generated as the sulphomethate substituents were released. Clinical resistance of P. aeruginosa to colomycin is rare, but a number of isolates have been isolated. Twelve colomycin-resistant clinical isolates were investigated to determine the mechanism of resistance. It was found that the low level of resistance was due to over expression of outer membrane protein H (OprH) in 5 isolates. A novel mechanism of resistance involving modification of the phosphate groups in LPS was identified in one of the isolates. Drugs which reduce inflammation in infected CF lungs would be of great advantage for therapy. Reducing inflammation would preserve the lung function and increase the quality of life for CF patients. Antibiotics like tetracyclines, macrolides and polymyxins were tested for their potential anti-inflammatory effects using cultured human monocytic (U937) cells which secrete the pro-inflammatory cytokines IL1- and TNF- in response to LPS from P. aeruginosa and B. cepacia. It was found that tetracyclines, and especially doxycycline, are good inhibitors of cytokine release by U937 cells and therefore could reduce the inflammatory cascade.

Development of a co-culture model of the human lungs for toxicity testing and identification of biomarkers of inhalation toxicity

The airway epithelium is the first point of contact in the lung for inhaled material, including infectious pathogens and particulate matter, and protects against toxicity from these substances by trapping and clearance via the mucociliary escalator, presence of a protective barrier with tight junctions and initiation of a local inflammatory response. The inflammatory response involves recruitment of phagocytic cells to neutralise and remove and invading materials and is oftern modelled using rodents. However, development of valid in vitro airway epithelial models is of great importance due to the restrictions on animal studies for cosmetic compound testing implicit in the 7th amendment to the European Union Cosmetics Directive. Further, rodent innate immune responses have fundamental differences to human. Pulmonary endothelial cells and leukocytes are also involved in the innate response initiated during pulmonary inflammation. Co-culture models of the airways, in particular where epithelial cells are cultured at air liquid interface with the presence of tight junctions and differentiated mucociliary cells, offer a solution to this problem. Ideally validated models will allow for detection of early biomarkers of response to exposure and investigation into inflammatory response during exposure. This thesis describes the approaches taken towards developing an in vitro epithelial/endothelial cell model of the human airways and identification biomarkers of response to exposure to xenobiotics. The model comprised normal human primary microvascular endothelial cells and the bronchial epithelial cell line BEAS-2B or normal human bronchial epithelial cells. BEAS-2B were chosen as their characterisation at air liquid interface is limited but they are robust in culture, thereby predicted to provide a more reliable test system. Proteomics analysis was undertaken on challenged cells to investigate biomarkers of exposure. BEAS-2B morphology was characterised at air liquid interface compared with normal human bronchial epithelial cells. The results indicate that BEAS-2B cells at an air liquid interface form tight junctions as shown by expression of the tight junction protein zonula occludens-1. To this author’s knowledge this is the first time this result has been reported. The inflammatory response of BEAS-2B (measured as secretion of the inflammatory mediators interleukin-8 and -6) air liquid interface mono-cultures to Escherichia coli lipopolysaccharide or particulate matter (fine and ultrafine titanium dioxide) was comparable to published data for epithelial cells. Cells were also exposed to polymers of “commercial interest” which were in the nanoparticle range (and referred to particles hereafter). BEAS-2B mono-cultures showed an increased secretion of inflammatory mediators after challenge. Inclusion of microvascular endothelial cells resulted in protection against LPS- and particle- induced epithelial toxicity, measured as cell viability and inflammatory response, indicating the importance of co-cultures for investigations into toxicity. Two-dimensional proteomic analysis of lysates from particle-challenged cells failed to identify biomarkers of toxicity due to assay interference and experimental variability. Separately, decreased plasma concentrations of serine protease inhibitors, and the negative acute phase proteins transthyretin, histidine-rich glycoprotein and alpha2-HS glycoprotein were identified as potential biomarkers of methyl methacrylate/ethyl methacrylate/butylacrylate treatment in rats.

The relationship between high-sensitivity CRP and polyclonal Free Light Chains as markers of inflammation in chronic disease

Introduction: Serum concentrations of polyclonal free light chains (FLC) represent the activity of the adaptive immune system. This study assessed the relationship between polyclonal FLC and the established marker of innate immunity, C-reactive protein (CRP), in chronic and acute disease. Methods: We utilized four cross-sectional chronic disease patient cohorts: chronic kidney disease (CKD), diabetes, vasculitis and kidney transplantation; and a longitudinal intensive care case series to assess the kinetics of production in acute disease. Results: There was a weak association between polyclonal FLC and high-sensitivity CRP (hs-CRP) in the study cohorts. A longitudinal assessment in acute disease showed a gradual increase in FLC concentrations over time, often when CRP levels were falling, demonstrating clear differences in the response kinetics of CRP and FLC in this setting. Conclusion: Polyclonal FLC and hs-CRP provide independent information as to inflammatory status. Prospective studies are now required to assess the utility of hs-CRP and polyclonal FLC in combination for risk stratification in disease populations. © 2013 John Wiley & Sons Ltd.

Vascular endothelial growth factor promotes physical wound repair and is anti-apoptotic in primary distal lung epithelial and A549 cells

Objective: There is evidence to suggest a beneficial role for growth factors, including vascular endothelial growth factor (VEGF), in tissue repair and proliferation after injury within the lung. Whether this effect is mediated predominantly by actions on endothelial cells or epithelial cells is unknown. This study tested the hypothesis that VEGF acts as an autocrine trophic factor for human adult alveolar epithelial cells and that under situations of pro-apoptotic stress, VEGF reduces cell death. Design: In vitro cell culture study looking at the effects of 0.03% H2O2 on both A549 and primary distal lung epithelial cells.Measurement and Main Results: Primary adult human distal lung epithelial cells express both the soluble and membrane-associated VEGF isoforms and VEGF receptors 1 and 2. At physiologically relevant doses, soluble VEGF isoforms stimulate wound repair and have a proliferative action. Specific receptor ligands confirmed that this effect was mediated by VEGF receptor 1. In addition to proliferation, we demonstrate that VEGF reduces A549 and distal lung epithelial cell apoptosis when administered after 0.03% H2O2 injury. This effect occurs due to reduced caspase-3 activation and is phosphatidylinositol 3′–kinase dependent. Conclusion: In addition to its known effects on endothelial cells, VEGF acts as a growth and anti-apoptotic factor on alveolar epithelial cells. VEGF treatment may have potential as a rescue therapy for diseases associated with alveolar epithelial damage such as acute respiratory distress syndrome.

Effects of recombinant human DNase and hypertonic saline on airway inflammation in children with cystic fibrosis

Recombinant human DNase (rhDNase) is an established treatment in cystic fibrosis (CF), but it may liberate cationic mediators bound to DNA in the airways. An alternative mucolytic therapy is hypertonic saline (HS); however, HS may potentiate neutrophilic inflammation. We compared the effect of rhDNase and HS on cationic proinflammatory mediators in CF sputum. In a randomized, crossover trial, 48 children with CF were allocated consecutively to 12 weeks of once-daily 2.5 mg rhDNase, alternate-day 2.5 mg rhDNase, and twice-daily 7% HS. Sputum levels of total interleukin-8 (IL-8), free IL-8, myeloperoxidase, eosinophil cationic protein, and neutrophil elastase (NE) activity were measured before and after each treatment. The change in mediator levels from baseline with daily rhDNase and HS was not significant; however, with alternate-day rhDNase, there was an increase in free IL-8. When changes in mediator levels with daily rhDNase were compared with alternate-day rhDNase and HS, no significant differences were detected. Only changes in NE activity were associated with changes in lung function. In summary, we were unable to show that rhDNase or HS promote airway inflammation in CF.