Christopher J. Nile

Expression and regulation of interleukin-33 in human monocytes

Interleukin‐33 (IL‐33) is an IL‐1 family cytokine that has a role in regulating T helper type 2 cytokines and mast cell development. Expression of IL‐33 is also associated with chronic inflammatory conditions such as rheumatoid arthritis. However, there is little information regarding IL‐33 in myeloid cell immune responses, which are important in immunity and inflammation. We therefore investigated the expression, intracellular location and regulation of myeloid cell IL‐33 by lipopolysaccharide (LPS) from Escherichia coli and the periodontal pathogen Porphyromonas gingivalis. We detected IL‐33 messenger RNA in the human promonocytic cell line THP‐1, in monocytes derived from these cells and in primary human monocytes. However, IL‐33 was not expressed in primary monocyte‐derived dendritic cells. Stimulation of monocytes with E. coli LPS (Toll‐like receptor 4 agonist) and LPS from P. gingivalis (Toll‐like receptor 2 agonist) up‐regulated IL‐33 at both the messenger RNA and protein levels but IL‐1β and tumour necrosis factor‐α had no effect. The IL‐33 protein was mainly found in the cytoplasm of monocytes with no evidence of nuclear translocation in stimulated cells. Furthermore, no IL‐33 secretion was detected after stimulation with LPS and/or ATP. These data indicate that the function, if any, of IL‐33 in activated monocytes is primarily intracellular. Interestingly, immunofluorescence analysis indicated that IL‐33 was sequestered in the nucleus of monocytes undergoing apoptosis but released into the extracellular milieu by LPS‐stimulated cells in which necrosis had been induced by freeze–thawing. Therefore, this endorses the view that IL‐33 may function as an ‘alarmin’ and have a role in signalling cellular damage and inflammatory disease pathogenesis through release from damaged or necrotic cells.

Differential expression of immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and Escherichia coli lipopolysaccharide

Porphyromonas gingivalis lipopolysaccharide (LPS) (strain W50) interacts with Toll‐like receptor 2 (TLR‐2) leading to cytokine expression and inflammation, and thereby plays a key role in the pathogenesis of periodontal disease. The aims of this study were to investigate gene expression of key regulatory mediators of innate immune responses in a human monocytic cell line (THP‐1) to P. gingivalis LPS and to compare these results with those obtained using the TLR‐4 ligand, Escherichia coli LPS. Custom‐made Taqman low‐density arrays were used for expression profiling of 45 different cytokine‐related genes. Both types of LPS highly up‐regulated interleukin (IL)‐1α and IL‐1β, IL‐18 receptor (IL‐18R), IL‐18R accessory protein and IL‐1 family (IL‐1F)9. Expression levels of IL‐1F6, IL‐1F7 and caspase‐1 were unaltered by either LPS. Genes for tumour necrosis factor‐α, IL‐6, leukaemia inhibitory factor and IL‐32 were also highly induced by both LPS. For a subset of genes, including CXC chemokine ligand 5 (CXCL5), expression was induced only by E. coli LPS or was up‐regulated more highly by E. coli compared with P. gingivalis LPS in THP‐1 monocytes. A similar expression pattern was also observed in dendritic cells. Analysis of signalling pathways which lead to CXCL5 expression indicated that the mechanisms underpinning the differential responses did not involve the recruitment of different adaptor proteins by TLR‐2 and TLR‐4, and therefore occur downstream of the receptor–adaptor complex. We conclude that differences in signalling pathways activated by TLR‐2 and TLR‐4 ligands lead to differential innate immune responses which may be important in polymicrobial diseases such as periodontal disease.

Biofilm formation is a risk factor for mortality in patients with Candida albicans bloodstream infection—Scotland, 2012–2013

Bloodstream infections caused by Candida species remain a significant cause of morbidity and mortality in hospitalized patients. Biofilm formation by Candida species is an important virulence factor for disease pathogenesis. A prospective analysis of patients with Candida bloodstream infection (n = 217) in Scotland (2012–2013) was performed to assess the risk factors associated with patient mortality, in particular the impact of biofilm formation. Candida bloodstream isolates (n = 280) and clinical records for 157 patients were collected through 11 different health boards across Scotland. Biofilm formation by clinical isolates was assessed in vitro with standard biomass assays. The role of biofilm phenotype on treatment efficacy was also evaluated in vitro by treating preformed biofilms with fixed concentrations of different classes of antifungal. Available mortality data for 134 patients showed that the 30-day candidaemia case mortality rate was 41%, with predisposing factors including patient age and catheter removal. Multivariate Cox regression survival analysis for 42 patients showed a significantly higher mortality rate for Candida albicans infection than for Candida glabrata infection. Biofilm-forming ability was significantly associated with C. albicans mortality (34 patients). Finally, in vitro antifungal sensitivity testing showed that low biofilm formers and high biofilm formers were differentially affected by azoles and echinocandins, but not by polyenes. This study provides further evidence that the biofilm phenotype represents a significant clinical entity, and that isolates with this phenotype differentially respond to antifungal therapy in vitro. Collectively, these findings show that greater clinical understanding is required with respect to Candida biofilm infections, and the implications of isolate heterogeneity.

Induction of Cationic Chicken Liver-Expressed Antimicrobial Peptide 2 in Response to Salmonella enterica Infection

ABSTRACT Cationic antimicrobial peptides constitute part of the innate immune system and provide an essential role in the defense against infection. At present there is a paucity of information regarding the antimicrobial profile of the chicken (Gallus gallus). Using in silico studies, an expressed sequence tag (EST) clone was identified which encodes a novel cationic antimicrobial peptide, chicken liver-expressed antimicrobial peptide 2 (cLEAP-2). The predicted amino acid sequence composed a prepropeptide, and the active peptide contained four conserved cysteine amino acids. The gene was localized to chromosome 13, and analysis of the genome revealed three exons separated by two introns. The cLEAP-2 gene was expressed in a number of chicken epithelial tissues including the small intestine, liver, lung, and kidney. Northern analysis identified liver-specific cLEAP-2 splice variants, suggesting some degree of tissue-specific regulation. To investigate whether cLEAP-2 expression was constitutive or induced in response to microbial infection, 4-day-old birds were orally infected with Salmonella. Analyses of cLEAP-2 expression by semiquantitative reverse transcription-PCR indicated that cLEAP-2 mRNA was upregulated significantly in the small intestinal tissues and the liver, indicative of direct and systemic responses. The antimicrobial activity of cLEAP-2 against Salmonella was analyzed in vitro with a time-kill assay and recombinant cLEAP-2. Interestingly Salmonella enterica serovar Typhimurium SL1344 showed increased susceptibility to the active cationic peptide (amino acids 37 to 76) compared to S. enterica serovar Typhimurium C5 and Salmonella enteritidis. Taken together, these data suggest that cationic cLEAP-2 is part of the innate host defense mechanisms of the chicken.

Leptin enhances the secretion of interleukin (IL)-18, but not IL-1β, from human monocytes via activation of caspase-1

Circulating levels of leptin are elevated in type-2 diabetes mellitus (T2DM) and leptin plays a role in immune responses. Elevated circulating IL-18 levels are associated with clinical complications of T2DM. IL-18 regulates cytokine secretion and the function of a number of immune cells including T-cells, neutrophils and macrophages and as such has a key role in immunity and inflammation. Pro-inflammatory monocytes exhibiting elevated cytokine secretion are closely associated with inflammation in T2DM, however, little is known about the role of leptin in modifying monocyte IL-18 secretion. We therefore aimed to investigate the effect of leptin on IL-18 secretion by monocytes. We report herein that leptin increases IL-18 secretion in THP-1 and primary human monocytes but has no effect on IL-18mRNA. Leptin and LPS signalling in monocytes occurs by overlapping but distinct pathways. Thus, in contrast to a strong stimulation by LPS, leptin has no effect on IL-1βmRNA levels or IL-1β secretion. In addition, LPS stimulates the secretion of IL-6 but leptin did not whereas both treatments up regulate IL-8 secretion from the same cells. Although leptin (and LPS) has a synergistic effect with exogenous ATP on IL-18 secretion in both THP-1 and primary monocytes, experiments involving ATP assays and pharmacological inhibition of ATP signalling failed to provide any evidence that endogenous ATP secreted by leptin-stimulated monocytes was responsible for enhancement of monocyte IL-18 secretion by leptin. Analysis of the action of caspase-1 revealed that leptin up regulates caspase-1 activity and the effect of leptin on IL-18 release is prevented by caspase-1 inhibitor (Ac-YVAD-cmk). These data suggest that leptin activates IL-18 processing rather than IL-18 transcription. In conclusion, leptin enhances IL-18 secretion via modulation of the caspase-1 inflammasome function and acts synergistically with ATP in this regard. This process may contribute to aberrant immune responses in T2DM and other conditions of hyperleptinemia.

The Oral Microbiome of Denture Wearers Is Influenced by Levels of Natural Dentition

Objectives The composition of dental plaque has been well defined, whereas currently there is limited understanding of the composition of denture plaque and how it directly influences denture related stomatitis (DS). The aims of this study were to compare the microbiomes of denture wearers, and to understand the implications of these towards inter-kingdom and host-pathogen interactions within the oral cavity. Methods Swab samples were obtained from 123 participants wearing either a complete or partial denture; the bacterial composition of each sample was determined using bar-coded illumina MiSeq sequencing of the bacterial hypervariable V4 region of 16S rDNA. Sequencing data processing was undertaken using QIIME, clustered in Operational Taxonomic Units (OTUs) and assigned to taxonomy. The dentures were sonicated to remove the microbial flora residing on the prosthesis, sonicate was then cultured using diagnostic colorex Candida media. Samples of unstimulated saliva were obtained and antimicrobial peptides (AMP) levels were measured by ELISA. Results We have shown that dental and denture plaques are significantly distinct both in composition and diversity and that the oral microbiome composition of a denture wearer is variable and is influenced by the location within the mouth. Dentures and mucosa were predominantly made up of Bacilli and Actinobacteria. Moreover, the presence of natural teeth has a significant impact on the overall microbial composition, when compared to the fully edentulous. Furthermore, increasing levels of Candida spp. positively correlate with Lactobacillus spp. AMPs were quantified, though showed no specific correlations. Conclusions This is the first study to provide a detailed understanding of the oral microbiome of denture wearers and has provided evidence that DS development is more complex than simply a candidal infection. Both fungal and bacterial kingdoms clearly play a role in defining the progression of DS, though we were unable to show a defined role for AMPs.

Polymicrobial Candida biofilms: friends and foe in the oral cavity

The role of polymicrobial biofilm infections in medicine is becoming more apparent. Increasing number of microbiome studies and deep sequencing has enabled us to develop a greater understanding of how positive and negative microbial interactions influence disease outcomes. An environment where this is particularly pertinent is within the oral cavity, a rich and diverse ecosystem inhabited by both bacteria and yeasts, which collectively occupy and coexist within various niches as biofilm communities. Studies within this environment have however tended to be subject to extensive independent investigation, in the context of either polymicrobial bacterial communities or yeast biofilms, but rarely both together. It is clear however that they are not mutually exclusive. Therefore, this review aims to explore the influence of candidal populations on the composition of these complex aggregates and biofilm communities, to investigate their mechanistic interactions to understand how these impact clinical outcomes, and determine whether we can translate how this knowledge can be used to improve patient management.

Gingival crevicular fluid, serum levels of receptor activator of nuclear factor-κB ligand, osteoprotegerin, and interleukin-17 in patients with rheumatoid arthritis and osteoporosis and with periodontal disease.

BACKGROUND This study is performed to evaluate gingival crevicular fluid (GCF) and serum levels of soluble receptor activator of nuclear factor-κB ligand (sRANKL), interleukin (IL)-17A, IL-17E, IL-17F, IL-17A/F, and osteoprotegerin (OPG) in women with rheumatoid arthritis (RA), osteoporosis (OPR), and those who are systemically healthy (SH), all with periodontal disease. METHODS GCF and serum samples were obtained before any periodontal intervention from 17 women with RA, 19 with OPR, and 13 who were SH with periodontitis. Full-mouth clinical periodontal measurements were recorded. sRANKL, OPG, and IL-17 levels were determined by enzyme-linked immunosorbent assay. RESULTS Clinical periodontal measurements were similar in the three study groups. Although the total amounts of GCF albumin, OPG, IL-17A, and IL-17A/F were similar in the study groups, there were statistically significant differences in GCF concentrations of sRANKL, OPG, IL-17A, IL-17E, IL-17F, and IL-17A/F. The sRANKL/OPG ratios were significantly higher in the RA group than in the OPR and SH groups (P <0.05). Serum sRANKL, sRANKL/OPG, and IL-17A/IL-17E ratios were significantly higher, whereas OPG concentrations were significantly lower in the RA group compared to other groups (P <0.05). Serum IL-17A concentrations were significantly higher in the RA and OPR groups than in the SH group (P <0.05). CONCLUSION Increased inflammatory mediator levels in patients with RA, despite the long-term use of various anti-inflammatory drugs, suggest that these patients may have a propensity to overproduce these inflammatory mediators.

Clinical associations between IL-17 family cytokines and periodontitis and potential differential roles for IL-17A and IL-17E in periodontal immunity

ObjectiveIL-17A is implicated in periodontitis pathogenesis. The roles of IL-17B–IL-17F and IL-17A/F are unknown. This study aimed to determine clinical associations between IL-17 family cytokines and periodontitis and to investigate the biological roles of IL-17A and IL-17E using in vitro model systems.Materials and methodsSamples from 97 patients with periodontitis and 77 healthy volunteers were used in the study. Serum, saliva and gingival crevicular fluid (GCF) levels of IL-17 family cytokines were measured by ELISA. Oral keratinocytes were stimulated with a P. gingivalis biofilm, or IL-17A, in the presence and absence of IL-17E and the expression of IL-8 and CXCL5 were investigated by ELISA and real-time-PCR. NF-κB phosphorylation in similar experiments was also measured using a cell-based ELISA.ResultsSerum, saliva and GCF IL-17A levels were higher in periodontitis patients and correlated positively with clinical parameters of attachment loss, pocket depth and bleeding on probing. Serum IL-17E levels were lower in periodontitis patients and the serum IL-17A:IL-17E ratio correlated positively with clinical parameters. In vitro, IL-17E inhibited Porphyromonas gingivalis and IL-17A induced expression of chemokines by reducing phosphorylation of the NF-κB p65 subunit.ConclusionsSerum IL-17A:IL-17E may be a marker of disease severity. IL-17E may have opposing roles to IL-17A in periodontitis pathogenesis. IL-17E can negatively regulate IL-17A and periodontal pathogen induced expression of chemokines by oral keratinocytes.

Stretch independent regulation of prostaglandin E(2) production within the isolated guinea-pig lamina propria.

To use an isolated preparation of the guinea‐pig bladder lamina propria (LP) to investigate the effects of adenosine tri‐phosphate (ATP) and nitric oxide (NO) on the release of prostaglandin E2 (PGE2).