TNF promoter hypomethylation is associated with mucosal inflammation in IBD and anti-TNF response.

Background and aims: Inflammatory Bowel Diseases (IBD) are chronic inflammatory conditions influenced heavily by environmental factors. DNA methylation is a form of epigenetic regulation linking environmental stimuli to gene expression changes and inflammation. Here, we investigated how DNA methylation of the TNF promoter differs between inflamed and uninflamed mucosa of IBD patients, including anti-TNF responders and non-responders. Methods: We obtained mucosal biopsies from 200 participants (133 IBD and 67 controls) and analyzed TNF promoter methylation using bisulfite sequencing, comparing inflamed with uninflamed segments, in addition to paired inflamed/uninflamed samples from individual patients. We conducted similar analyses on purified intestinal epithelial cells from bowel resections. We also compared TNF methylation levels of inflamed and uninflamed mucosa from a separate cohort of 15 anti-TNF responders and 17 non-responders. Finally, we sequenced DNA methyltransferase genes to identify rare variants in IBD patients and functionally tested them using rescue experiments in a zebrafish genetic model of DNA methylation deficiency. Results: TNF promoter methylation levels were decreased in inflamed mucosa of IBD patients and correlated with disease severity. Isolated IECs from inflamed tissue showed proportional decreases in TNF methylation. Anti-TNF non-responders showed lower levels of TNF methylation than responders in uninflamed mucosa. Our sequencing analysis revealed two missense variants in DNMT1, one of which had reduced function in vivo. Conclusions: Our study reveals an association of TNF promoter hypomethylation with mucosal inflammation, suggesting that IBD patients may be particularly sensitive to inflammatory environmental insults affecting DNA methylation. Together, our analyses indicate that TNF promoter methylation analysis may aid in the characterization of IBD status and evaluation of anti-TNF therapy response.

AAV-mediated expression of HLA-G for the prevention of experimental ocular graft vs. host disease.

Ocular graft versus host disease (OGvHD) develops after allogeneic hematopoietic stem cell transplantation (HSCT) and manifests as ocular surface inflammatory disease. This study evaluated the efficacy of adeno-associated virus (AAV) gene therapy encoding human leukocyte antigen G (HLA-G) to inhibit OGvHD. A major histocompatibility mismatch chronic OGvHD murine model was evaluated. 7 days after HSCT, mice were dosed subconjunctivally with scAAV8-HLA-G1/5 (1 x 109 vg/eye), topical cyclosporine (twice daily), or left untreated. Body weights and tear production (red thread test) were recorded, and eyelid, corneal opacity, and corneal fluorescein retention were scored through day 44 after HSCT. Tissues were collected for vector biodistribution, ocular histology, and immunofluorescence. Compared with untreated HSCT eyes, those dosed with scAAV8-HLA-G1/5 had significantly reduced clinical inflammatory signs of OGvHD. On histology, eyes that received scAAV8-HLA-G1/5 or cyclosporine had a significantly lower mean limbal mononuclear cell count when compared with non-treated HSCT eyes. HLA-G immunofluorescence was detected in the subconjunctiva and peripheral cornea in HSCT animals treated with scAAV8-HLA-G1/5. Vector genomes were detected in the lacrimal gland, but not in the other tested organs. These results provide evidence that subconjunctival AAV targets ocular surface and corneal disease and support that HLA-G-based gene therapy may be an effective treatment for OGvHD.

Distinct roles for luminal acidification in apical protein sorting and trafficking in zebrafish.

Epithelial cell physiology critically depends on the asymmetric distribution of channels and transporters. However, the mechanisms targeting membrane proteins to the apical surface are still poorly understood. Here, we performed a visual forward genetic screen in the zebrafish intestine and identified mutants with defective apical targeting of membrane proteins. One of these mutants, affecting the vacuolar H+-ATPase gene atp6ap1b, revealed specific requirements for luminal acidification in apical, but not basolateral, membrane protein sorting and transport. Using a low temperature block assay combined with genetic and pharmacologic perturbation of luminal pH, we monitored transport of newly synthesized membrane proteins from the TGN to apical membrane in live zebrafish. We show that vacuolar H+-ATPase activity regulates sorting of O-glycosylated proteins at the TGN, as well as Rab8-dependent post-Golgi trafficking of different classes of apical membrane proteins. Thus, luminal acidification plays distinct and specific roles in apical membrane biogenesis.

TNFa/TNFR2 signaling is required for glial ensheathment at the dorsal root entry zone.

Somatosensory information from the periphery is routed to the spinal cord through centrally-projecting sensory axons that cross into the central nervous system (CNS) via the dorsal root entry zone (DREZ). The glial cells that ensheath these axons ensure rapid propagation of this information. Despite the importance of this glial-axon arrangement, how this afferent nerve is assembled during development is unknown. Using in vivo, time-lapse imaging we show that as centrally-projecting pioneer axons from dorsal root ganglia (DRG) enter the spinal cord, they initiate expression of the cytokine TNFalpha. This induction coincides with ensheathment of these axons by associated glia via a TNF receptor 2 (TNFR2)-mediated process. This work identifies a signaling cascade that mediates peripheral glial-axon interactions and it functions to ensure that DRG afferent projections are ensheathed after pioneer axons complete their navigation, which promotes efficient somatosensory neural function.

Infection, Inflammation and Healing in Zebrafish: Intestinal Inflammation.

Inflammatory bowel diseases (IBD), which include Crohn's disease and ulcerative colitis, contribute to significant morbidity and mortality globally. Despite an increase in incidence, IBD onset is still poorly understood. Mouse models of IBD recapitulate several aspects of human disease, but limited accessibility for live imaging and the lack of forward genetics highlight the need for new model systems for disease onset characterization. Zebrafish represent a powerful platform to model IBD using forward and reverse genetics, live imaging of transgenic lines and physiological assays. In this review, we address current models of IBD in zebrafish and newly developed reagents available for future studies.

Epigenetic control of intestinal barrier function and inflammation in zebrafish.

The intestinal epithelium forms a barrier protecting the organism from microbes and other proinflammatory stimuli. The integrity of this barrier and the proper response to infection requires precise regulation of powerful immune homing signals such as tumor necrosis factor (TNF). Dysregulation of TNF leads to inflammatory bowel diseases (IBD), but the mechanism controlling the expression of this potent cytokine and the events that trigger the onset of chronic inflammation are unknown. Here, we show that loss of function of the epigenetic regulator ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1) in zebrafish leads to a reduction in tnfa promoter methylation and the induction of tnfa expression in intestinal epithelial cells (IECs). The increase in IEC tnfa levels is microbe-dependent and results in IEC shedding and apoptosis, immune cell recruitment, and barrier dysfunction, consistent with chronic inflammation. Importantly, tnfa knockdown in uhrf1 mutants restores IEC morphology, reduces cell shedding, and improves barrier function. We propose that loss of epigenetic repression and TNF induction in the intestinal epithelium can lead to IBD onset.

Rapid identification of kidney cyst mutations by whole exome sequencing in zebrafish.

Forward genetic approaches in zebrafish have provided invaluable information about developmental processes. However, the relative difficulty of mapping and isolating mutations has limited the number of new genetic screens. Recent improvements in the annotation of the zebrafish genome coupled to a reduction in sequencing costs prompted the development of whole genome and RNA sequencing approaches for gene discovery. Here we describe a whole exome sequencing (WES) approach that allows rapid and cost-effective identification of mutations. We used our WES methodology to isolate four mutations that cause kidney cysts; we identified novel alleles in two ciliary genes as well as two novel mutants. The WES approach described here does not require specialized infrastructure or training and is therefore widely accessible. This methodology should thus help facilitate genetic screens and expedite the identification of mutants that can inform basic biological processes and the causality of genetic disorders in humans.

Cftr controls lumen expansion and function of Kupffer's vesicle in zebrafish.

Regulated fluid secretion is crucial for the function of most organs. In vertebrates, the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) is a master regulator of fluid secretion. Although the biophysical properties of CFTR have been well characterized in vitro, little is known about its in vivo role during development. Here, we investigated the function of Cftr during zebrafish development by generating several cftr mutant alleles using TAL effector nucleases. We found that loss of cftr function leads to organ laterality defects. In zebrafish, left-right (LR) asymmetry requires cilia-driven fluid flow within the lumen of Kupffer's vesicle (KV). Using live imaging we found that KV morphogenesis is disrupted in cftr mutants. Loss of Cftr-mediated fluid secretion impairs KV lumen expansion leading to defects in organ laterality. Using bacterial artificial chromosome recombineering, we generated transgenic fish expressing functional Cftr fusion proteins with fluorescent tags under the control of the cftr promoter. The transgenes completely rescued the cftr mutant phenotype. Live imaging of these transgenic lines showed that Cftr is localized to the apical membrane of the epithelial cells in KV during lumen formation. Pharmacological stimulation of Cftr-dependent fluid secretion led to an expansion of the KV lumen. Conversely, inhibition of ion gradient formation impaired KV lumen inflation. Interestingly, cilia formation and motility in KV were not affected, suggesting that fluid secretion and flow are independently controlled in KV. These findings uncover a new role for cftr in KV morphogenesis and function during zebrafish development.

Rapid differential transport of Nodal and Lefty on sulfated proteoglycan-rich extracellular matrix regulates left-right asymmetry in Xenopus.

The spatiotemporally dynamic distribution of instructive ligands within embryonic tissue, and their feedback antagonists, including inherent stabilities and rates of clearance, are affected by interactions with cell surfaces or extracellular matrix (ECM). Nodal (here, Xnr1 or Nodal1 in Xenopus) and Lefty interact in a cross-regulatory relationship in mesendoderm induction, and are the conserved instructors of left-right (LR) asymmetry in early somitogenesis stage embryos. By expressing Xnr1 and Lefty proproteins that produce mature functional epitope-tagged ligands in vivo, we found that ECM is a principal surface of Nodal and Lefty accumulation. We detected Lefty moving faster than Nodal, with evidence that intact sulfated proteoglycans in the ECM facilitate the remarkable long distance movement of Nodal. We propose that Nodal autoregulation substantially aided by rapid ligand transport underlies the anteriorward shift of Nodal expression in the left LPM (lateral plate mesoderm), and speculate that the higher levels of chondroitin-sulfate proteoglycan (CSPG) in more mature anterior regions provide directional transport cues. Immunodetection and biochemical analysis showed transfer of Lefty from left LPM to right LPM, providing direct evidence that left-side-derived Lefty is a significant influence in ensuring the continued suppression of right-sided expression of Nodal, maintaining unilateral expression of this conserved determinant of asymmetry.