The TLR4 D299G and T399I SNPs Are Constitutively Active to Up-Regulate Expression of Trif-Dependent Genes

Peer reviewedPublisher PD

The microaerophilic microbiota of de-novo paediatric inflammatory bowel disease: the BISCUIT study

<p>Introduction: Children presenting for the first time with inflammatory bowel disease (IBD) offer a unique opportunity to study aetiological agents before the confounders of treatment. Microaerophilic bacteria can exploit the ecological niche of the intestinal epithelium; Helicobacter and Campylobacter are previously implicated in IBD pathogenesis. We set out to study these and other microaerophilic bacteria in de-novo paediatric IBD.</p> <p>Patients and Methods: 100 children undergoing colonoscopy were recruited including 44 treatment naïve de-novo IBD patients and 42 with normal colons. Colonic biopsies were subjected to microaerophilic culture with Gram-negative isolates then identified by sequencing. Biopsies were also PCR screened for the specific microaerophilic bacterial groups: Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis.</p> <p>Results: 129 Gram-negative microaerophilic bacterial isolates were identified from 10 genera. The most frequently cultured was S. wadsworthensis (32 distinct isolates). Unusual Campylobacter were isolated from 8 subjects (including 3 C. concisus, 1 C. curvus, 1 C. lari, 1 C. rectus, 3 C. showae). No Helicobacter were cultured. When comparing IBD vs. normal colon control by PCR the prevalence figures were not significantly different (Helicobacter 11% vs. 12%, p = 1.00; Campylobacter 75% vs. 76%, p = 1.00; S. wadsworthensis 82% vs. 71%, p = 0.312).</p> <p>Conclusions: This study offers a comprehensive overview of the microaerophilic microbiota of the paediatric colon including at IBD onset. Campylobacter appear to be surprisingly common, are not more strongly associated with IBD and can be isolated from around 8% of paediatric colonic biopsies. S. wadsworthensis appears to be a common commensal. Helicobacter species are relatively rare in the paediatric colon.</p&gt

Role of host genetics in fibrosis

Fibrosis can occur in tissues in response to a variety of stimuli. Following tissue injury, cells undergo transformation or activation from a quiescent to an activated state resulting in tissue remodelling. The fibrogenic process creates a tissue environment that allows inflammatory and matrix-producing cells to invade and proliferate. While this process is important for normal wound healing, chronicity can lead to impaired tissue structure and function

Functional linkage of gene expression patterns.

<p>(A) Representation of gene expression patterns affected by the TLR4 Asp299Gly and Thr399Ile polymorphisms in unstimulated PBMCs using Ingenuity pathway analysis. A log₂ ratio cutoff of 1 was set to focus on genes with greater than 2-fold differential regulation between the two groups. Intensity of colouring indicates the strength of the up-regulation (strong green, highly up-regulated in TLR4 wildtype subjects; strong red, highly upregulated in TLR4 polymorphic subjects; no colour indicates genes included to provide completeness of pathways). (B) Hierarchical clustering analysis of selected gene regulation by the TLR4 Asp299Gly and Thr399Ile polymorphisms in PBMCs following 2 hr LPS stimulation. All genes (n = 41) that following normalisation to housekeeper gene showed down-regulation in polymorphic samples compared to wildtype under LPS stimulation. The columns depict individual subjects (1–6), C =  TLR4 wildtype subjects, M =  TLR4 polymorphic (Asp299Gly and Thr399Ile) subjects. Each row represents a single gene, the key on LHS represents the range of expression level values from the data displayed in the heatmap.</p

Transient transfection of TLR4 Asp299Gly, TLR4 Thr399Ile or TLR4 Asp299Gly/Thr399Ile with MD2 into HEK cells results in constitutive NF-κB activation but a reduced relative increase compared to basal NF-κB activity.

<p>HEK cells were transiently transfected with wild-type or mutant TLR4 +MD-2 +CD14, together with the reporter constructs NF-κB-luc and Renilla luciferase. After 48 h the cells were stimulated for 6 h with medium alone (C) or medium +10 ng/ml LPS (LPS) and the luciferase activity was determined in cell lysates. Data are from a representative experiment (n = 3 experiments) and are shown as mean ±SEM for that experiment. A) Relative luciferase activity of wild type TLR4, Asp299Gly TLR4, Thr399Ile TLR4 or Asp299Gly/Thr399Ile TLR4 with or without stimulation with 10 ng/ml LPS. B) Fold increase in luciferase activity compared to basal activity induced by stimulation of wild type TLR4, Asp299Gly TLR4, Thr399Ile TLR4 or Asp299Gly/Thr399Ile TLR4 with 10 ng/ml LPS. C MD2  =  unstimulated cells in the presence of MD2; LPS MD2  =  LPS stimulated cells in the presence of MD2; C-MD2  =  unstimulated cells with MD2 absent; LPS-MD2  =  LPS stimulated cells with MD2 absent.</p