Expression profile of the matricellular protein periostin in paediatric inflammatory bowel disease

The precise role of periostin, an extra-cellular matrix protein, in inflammatory bowel disease (IBD) is unclear. Here, we investigated periostin in paediatric IBD including its relationship with disease activity, clinical outcomes, genomic variation and expression in the colonic tissue. Plasma periostin was analysed using ELISA in 144 paediatric patients and 38 controls. Plasma levels were assessed against validated disease activity indices in IBD and clinical outcomes. An immuno-fluorescence for periostin and detailed isoform-expression analysis in the colonic tissue was performed in 23 individuals. We integrated a whole-gene based burden metric ‘GenePy’ to assess the impact of variation in POSTN and 23 other genes functionally connected to periostin. We found that plasma periostin levels were significantly increased during remission compared to active Crohn’s disease. The immuno-fluorescence analysis demonstrated enhanced peri-cryptal ring patterns in patients compared to controls, present throughout inflamed, as well as macroscopically non-inflamed colonic tissue. Interestingly, the pattern of isoforms remained unchanged during bowel inflammation compared to healthy controls. In addition to its role during the inflammatory processes in IBD, periostin may have an additional prominent role in mucosal repair. Additional studies will be necessary to understand its role in the pathogenesis, repair and fibrosis in IBD.

pending analysis. Anti-human periostin (abcam ab14041) were used as the primary antibodies and cy3-labeled anti-rabbit antibody (abcam ab6939) as the secondary antibodies.
The frozen block was removed from the -80 o C freezer, allowed to equilibrate in the cryostat chamber at -20 o C for approximately 30 minutes. Cryostat sectioning of the tissue (6-8 microns) was performed using anti-roll plate method. Slides were drop fixed in 4% paraformaldehyde (PFA) and phosphate buffer solution (PBS) for 5 minutes followed by a wash cycle in PBS. Fixatives preserve tissue architecture, inactivate proteolytic enzymes that could otherwise degrade the sample, stabilise specimens so that they can withstand further processing and protect samples against microbial contamination 2 . Slides were thereafter treated with PBT (PBS with 0.1% triton) for 10 minutes to allow intracellular staining. To block non-specific binding reactions, sections were incubated with foetal calf serum (50% FCS) in PBT for 1 hour at room temperature. This was followed by overnight incubation of the slides at 4 o C with the primary antibody (anti-human periostin) diluted in PBT with 10% FCS (1:500 dilution). After wash cycles with PBS, the slides were treated with secondary antibodies for 2 hours at room temperature and washed again 3 times for 5 minutes with PBS.
To the second wash DAPI was added for staining of nuclei. 5to 10 sections were analysed per sample. Analysis was done with Axiovert microscope.

Section 3. Periostin isoform analysis
RNA was isolated from intestinal biopsies of patients with ulcerative colitis and crohn's disease as well as non-IBD controls using Qiagen RNeasy kit. RNA was then reverse transcribed into cDNA, which was then used for PCR with one primer specific for Exon 16 (forward primer: CCTTCAAAGAAATCCCCGTGACTGTC) and one reverse primer specific for Exon 23 (reverse primer: TCACTGAGAACGACCTTCCCTTAATC). These primers detect all isoforms of periostin present in a given tissue, as they amplify the entire alternatively spliced area. The PCR mix was analysed by gel electrophoresis (agarose gels and agilent) and additionally cloned into plasmid pJET1.2/blunt using CloneJet PCR cloning kit (Thermofisher). Subsequently, plasmid DNAs were isolated and sequenced.

Section 4. DNA extraction
Genomic DNA was extracted from peripheral venous blood or saliva specimens using the salting out method 3 . For venous blood specimens, buffy coats of nucleated cells were subjected to cell and nuclear membrane lysis using nuclei lysis buffer (10 mM Tris-HCl, 400 mM NaCl and 2 Mm Na 2 EDTA, pH 8.2), cell lysates digested overnight at 37∘ C with 0.2 ml of 10% SDS and 0.5 ml of a protease K solution (1 mg protease K in 1% SDS and 2mM Na 2 EDTA), followed by addition of saturated sodium chloride and centrifugation at 2500 rpm for 15 minutes. The supernatant containing the DNA is then treated with absolute ethanol until DNA is precipitated, followed by transfer to micro-centrifuge tube containing 100-200µl TE buffer (10:10 mM Tris-HCl, 400 mM NaCl and 2 Mm Na 2 EDTA, pH 7.5).
The DNA is allowed to dissolve for 2 hours at 37∘ C, then quantified using the Qubit ® 2.0 Fluorometer and a 260:280 ratio calculated using a nanodrop spectrophotometer. The average DNA yield obtained is 150µg/ml and approximately 20ug of DNA is used for next generation sequencing for each patient.

Section 5. Processing of whole exome sequencing (WES) data
Genomic DNA was extracted from peripheral venous blood (see section 4) and fragmented DNA subjected adaptor ligation and exome library enrichment using the Agilent SureSelect All Exon capture kit versions 4, 5 and 6. Enriched libraries were sequenced on Illumina HiSeq systems. Alignment against the human genome (hg38) was performed using Burrows-Wheeler Aligner (BWA) 4 , variants called using Genome Analysis Toolkit (GATK v3.8) and ANNOVAR for variant annotation 5 . Section 6. WES data analysis using GenePy score WES data analysis was conducted using a whole gene-based pathogenicity score, 'GenePy' as previously described 6 . GenePy incorporates known deleteriousness metrics, allele frequency and individual zygosity information for each variant and sums them to generate a whole gene-based pathogenicity score. Each gene score was subsequently corrected for gene length. In this study, GenePy scores were generated for a list of genes selected for their functional relevance to periostin. For the selection of genes, an electronic search was conducted through 'PathCards', an online database of human biological pathways 7 .
PathCards generates a unified set of genes for the interrogated pathway or protein in the form of 'SuperPaths', consolidating biological information from multiple manually curated sources. In this study, the 'SuperPath' nucleating from the search by entering the term 'periostin' was used to select the genes for application of GenePy scores. Interrogation through PathCards using the search term 'periostin' displayed two SuperPaths, 'Amplification and Expansion of Oncogenic Pathways as Metastatic Traits' and 'Hypothesized Pathways in Pathogenesis of Cardiovascular Disease', each comprising seventeen and twenty-five genes respectively, including periostin. The former was selected based on its functional relevance to inflammatory pathologies, given the well-characterised roles of periostin in inflammatory and oncological diseases.
GenePy scores were available for twenty four out of the twenty-seven genes selected.
GenePy scores were not available for CCL5, ITGA5B1 and ITGA5B3 genes. Scores may not be available for all genes as some of the genes may not be properly captured during the exome library capture process, or may not be annotated by the currently available computational databases used when generating GenePy scores.