Key Points• Restricting transgenic antigen expression to differentiated antigen-presenting cells protects hematopoietic progenitors from immune attack.• Restricting transgenic antigen expression to differentiated antigen-presenting cells promotes tolerogenic outcomes.

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MicroRNAs (miRNAs) are short non-coding RNAs involved in biological and pathological processes of every cell type, including liver cells. Transcribed from specific genes, miRNA precursors are processed in the cytoplasm into mature miRNAs and as part of the RNA-induced silencing complex (RISC) complex binds to messenger RNA (mRNA) by imperfect complementarity. This leads to the regulation of gene expression at a post-transcriptional level. The function of a number of different miRNAs in fibrogenesis associated with the progression of chronic liver disease has recently been elucidated. Furthermore, miRNAs have been shown to be both disease-and tissue-specific and are stable in the circulation, which has led to increasing investigation on their utility as biomarkers for the diagnosis of chronic liver diseases, including those in children. Here, we review the current knowledge on the biogenesis of microRNA, the mechanisms of translational repression and the use of miRNA as circulatory biomarkers in chronic paediatric liver diseases including cystic fibrosis associated liver disease, biliary atresia and viral hepatitis B.

Islet-specific memory T cells arise early in type 1 diabetes (T1D), persist for long periods, perpetuate disease, and are rapidly reactivated by islet transplantation. As memory T cells are poorly controlled by "conventional" therapies, memory T cell-mediated attack is a substantial challenge in islet transplantation, and this will extend to application of personalized approaches using stem cell-derived replacement b-cells. New approaches are required to limit memory autoimmune attack of transplanted islets or replacement b-cells. Here, we show that transfer of bone marrow encoding cognate antigen directed to dendritic cells, under mild, immune-preserving conditions, inactivates established memory CD8 + T-cell populations and generates a long-lived, antigen-specific tolerogenic environment. Consequently, CD8 + memory T cell-mediated targeting of islet-expressed antigens is prevented and islet graft rejection alleviated. The immunological mechanisms of protection are mediated through deletion and induction of unresponsiveness in targeted memory T-cell populations. The data demonstrate that hematopoietic stem cell-mediated gene therapy effectively terminates antigenspecific memory T-cell responses, and this can alleviate destruction of antigen-expressing islets. This addresses a key challenge facing islet transplantation and, importantly, the clinical application of personalized b-cell replacement therapies using patient-derived stem cells.

Cystic fibrosis (CF)-associated liver disease (CFLD) is a hepatobiliary complication of CF. Current diagnostic modalities rely on nonspecific assessments, whereas liver biopsy is the gold standard to assess severity of fibrosis. MicroRNAs (miRNAs) regulate liver disease pathogenesis and are proposed as diagnostic biomarkers. We investigated the combined use of serum miRNAs and aspartate aminotransferase (AST) to platelet ratio (APRI) to diagnose and assess CFLD severity. This was a cross-sectional cohort study of the circulatory miRNA signature of 124 children grouped by clinical, biochemical, and imaging assessments as follows: CFLD (n = 44), CF patients with no evidence of liver disease (CFnoLD; n = 40), and healthy controls (n = 40). Serum miRNAs were analyzed using miRNA sequencing (miRNA-Seq). Selected differentially expressed serum miRNA candidates were further validated by qRT-PCR and statistical analysis performed to evaluate utility to predict CFLD and fibrosis severity validated by liver biopsy, alone or in combination with APRI. Serum miR-122-5p, miR-365a-3p, and miR-34a-5p levels were elevated in CFLD compared to CFnoLD, whereas miR-142-3p and let-7g-5p were down-regulated in CFLD compared to CFnoLD. Logistic regression analysis combining miR-365a-3p, miR-142-3p, and let-7g-5p with APRI showed 21 times greater odds of accurately predicting liver disease in CF with an area under the receiver operating characteristics curve (AUROC) = 0.91 (sensitivity = 83%, specificity = 92%; P < 0.0001). Expression levels of serum miR-18a-5p were correlated with increasing hepatic fibrosis (HF) stage in CFLD (r = 0.56; P < 0.0001), showing good diagnostic accuracy for distinguishing severe (F3-F4) from mild/moderate fibrosis (F0-F2). A unit increase of miR-18a-5p showed a 7-fold increased odds of having severe fibrosis with an AUROC = 0.82 (sensitivity = 93%, specificity = 73%; P = 0.004), indicating its potential to predict fibrosis severity. Conclusion: We identified a distinct circulatory miRNA profile in pediatric CFLD with potential to accurately discriminate liver disease and fibrosis severity in children with CF.

During chronic liver injury hepatic stellate cells (HSCs), the principal source of extracellular matrix in the fibrotic liver, transdifferentiate into pro-fibrotic myofibroblast-like cells - a process potentially regulated by microRNAs (miRNAs). Recently, we found serum miRNA-25-3p (miR-25) levels were upregulated in children with Cystic Fibrosis (CF) without liver disease, compared to children with CF-associated liver disease and healthy individuals. Here we examine the role of miR-25 in HSC biology. MiR-25 was detected in the human HSC cell line LX-2 and in primary murine HSCs, and increased with culture-induced activation. Transient overexpression of miR-25 inhibited TGF-β and its type 1 receptor (TGFBR1) mRNA expression, TGF-β-induced Smad2 phosphorylation and subsequent collagen1α1 induction in LX-2 cells. Pull-down experiments with biotinylated miR-25 revealed Notch signaling (co-)activators ADAM-17 and FKBP14 as miR-25 targets in HSCs. NanoString analysis confirmed miR-25 regulation of Notch- and Wnt-signaling pathways. Expression of Notch signaling pathway components and endogenous Notch1 signaling was downregulated in miR-25 overexpressing LX-2 cells, as were components of Wnt signaling such as Wnt5a. We propose that miR-25 acts as a negative feedback anti-fibrotic control during HSC activation by reducing the reactivity of HSCs to TGF-β-induced collagen expression and modulating the cross-talk between Notch, Wnt and TGF-β signaling.

Transplantation of hematopoietic stem cells (HSC) or bone marrow (BM) engineered to encode antigens is a means of inducing antigen-specific tolerance that might be suitable for ‘turning off’ pathogenic effector and memory T-cell responses. However, transplantation into primed hosts under non-myeloablative conditions could lead to rejection of engineered HSC or BM. We hypothesized targeting antigen expression to mature APC would alleviate rejection of engineered HSC while still permitting expression in the ‘tolerogenic’ cells that arise after transplantation. Using non-myeloablative conditions, we compared engraftment and ‘tolerogenicity’ in naïve or immune hosts of BM encoding an antigen that was ubiquitously expressed, targeted to diverse APC, or targeted to DC. When antigen was expressed ubiquitously, engraftment failed (&lt;1%) but targeting antigen to DC, permitted high levels of BM engraftment (≈40%). Unexpectedly, BM failed to engraft (&lt;1%) when antigen was targeted using a MHC class II promoter. This was due low level MHC class II expression by HSC leading to antigen expression and subsequent immune rejection of engrafting HSC. Long-term tolerance of antigen-specific memory T cells occurred only when engraftment was facilitated by targeting antigen to DC. For clinical translation, careful selection for a promoter that optimally targets DC will be imperative to ensure engraftment of antigen-encoding cells and long-term tolerance.