Transcriptome analysis of ankylosing spondylitis patients before and after TNF-α inhibitor therapy reveals the pathways affected.

Tumor necrosis factor-α (TNF-α) inhibitors are highly effective in suppressing inflammation in ankylosing spondylitis (AS) patients, and operate by suppression of TFN-α and downstream immunological pathways. To determine the mechanisms of action of TNF-α inhibitors in AS patients, we used transcriptomic and bioinformatic approaches on peripheral blood mononuclear cells from AS patients pre and post treatment. We found 656 differentially expressed genes, including the genome-wide significant AS-associated genes, IL6R, NOTCH1, IL10, CXCR2 and TNFRSF1A. A distinctive gene expression profile was found between male and female patients, mainly because of sex chromosome-linked genes and interleukin 17 receptor C, potentially accounting for the differences in clinical manifestation and treatment response between the genders. In addition to immune and inflammation regulatory pathways, like intestinal immune network for IgA production, cytokine-cytokine receptor interaction, Ras signaling pathway, allograft rejection and hematopoietic cell lineage, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses revealed that infection-associated pathways (influenza A and toxoplasmosis) and metabolism-associated pathways were involved in response to TNF-α inhibitor treatment, providing insight into the mechanism of TNF-α inhibitors.

Epigenetic and gene expression analysis of ankylosing spondylitis-associated loci implicate immune cells and the gut in the disease pathogenesis.

Ankylosing spondylitis (AS) is a common immune-mediated arthropathy primarily affecting the spine and pelvis. Most AS patients have subclinical intestinal inflammation, suggesting the gut microbiome and the immune response play a role in pathogenesis. Susceptibility to AS is primarily genetic, and at least 114 susceptibility variants have been identified to date. We applied bioinformatic methods utilizing epigenetic and gene and protein expression data to identify the cell types through which AS-associated variants operate. Variants were enriched in transcriptionally regulated regions in monocytes, CD4+ and CD8+ T cells, natural killer cells, regulatory T cells and B cells and mucosa from the small intestine, sigmoid colon and rectum. Weak signals were detected in bone cells, consistent with bone disease being a secondary manifestation. RNA sequencing of blood cells from AS patients and controls identified differentially expressed genes. Interrogation of expression databases showed that the upregulated genes were enriched in monocytes and downregulated genes were enriched in CD8+ T cells and natural killer cells. Gene Ontology term enrichment analysis identified microbes and the gut in the aetiology of AS. These findings identify the key immune cell types that drive the disease, and further highlight the involvement of the gut microbiome in the pathogenesis of AS.

Cloning and functional analysis of the Sry-related HMG box gene, Sox18.

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii) an intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor -2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue.

Crim1 has an essential role in glycogen trophoblast cell and sinusoidal-trophoblast giant cell development in the placenta.

Normal placental development and function is essential for fetal growth of eutherian mammals. Mutational studies have shown that numerous growth factors are required for placental development and differentiation of placental lineages. Here, using a gene-trap mutant mouse line, Crim1(KST264), we show that Crim1 is essential for murine placental development. Crim1 is a developmentally expressed, trans-membrane regulator of growth factor activity. Crim1(KST264/KST264) mutant placentae displayed hypoplasia from 13.5 dpc, and altered structure from 15.5 dpc, including alterations in cell number in both the junctional and labyrinth zones. Using the reporter gene from the Crim1(KST264) allele, we found that Crim1 is expressed in multiple cell types of the placenta, including strong expression in the spongiotrophoblast cells of the junctional zone. In the junctional zone of Crim1(KST264/KST264) placentae, there was an increase in the glycogen trophoblast cells adjacent to the spongiotrophoblast cells. In the labyrinth zone, we found a decrease in the density of sinusoidal-trophoblast giant cells. Our findings show that Crim1 is required for placental development, and is necessary for the proper differentiation of sinusoidal-trophoblast giant cells and glycogen trophoblast cells.

Targeted disruption of the Wnt2 gene results in placentation defects.

Wnt genes have been implicated in a range of developmental processes in the mouse including the patterning of the central nervous system and limbs. Reported here for the first time is the expression of Wnt2 in the early heart field of 7.5-8.5 dpc (days post-coitum) mouse embryos, making Wnt2 a potentially useful gene marker for the early stages of heart development. Expression was also detected in the allantois from 8.0 dpc and at later stages in the placenta and umbilicus. Mice deficient in Wnt2, generated by gene targeting, displayed runting and approximately 50% died perinatally. Histological analysis revealed alterations in the size and structure of placentas from these mice from 14.5 dpc. The placental defects were associated primarily with the labyrinthine zone and included oedema and tissue disruption and accumulation of maternal blood in large pools. There was also an apparent decrease in the number of foetal capillaries and an increase in the amount of fibrinoid material in the Wnt2 mutant placentas. These results suggest that Wnt2 is required for the proper vascularisation of the mouse placenta and the placental defects in Wnt2-deficient mice result in a reduction in birthweight and perinatal lethality.