Bacteria are thought to contribute to the pathogenesis of necrotizing enterocolitis (NEC), but it is unknown whether their interaction with the epithelium can participate in the initiation of mucosal injury or they can act only following translocation across a damaged intestinal barrier. Our aims were to determine whether bacteria and intestinal epithelial TLR4 play roles in a well-established neonatal rat model and a novel neonatal murine model of NEC. Neonatal rats, C57BL/6J, C3HeB/FeJ (TLR4 wild type), and C3H/HeJ (TLR4 mutant) mice were delivered by Cesarean section and were subjected to formula feeding and cold asphyxia stress or were delivered naturally and were mother-fed. NEC incidence was evaluated by histological scoring, and gene expression was quantified using quantitative real-time PCR from cDNA generated from intestinal total RNA or from RNA obtained by laser capture microdissection. Spontaneous feeding catheter colonization or supplementation of cultured bacterial isolates to formula increased the incidence of experimental NEC. During the first 72 h of life, i.e., the time frame of NEC development in this model, intestinal TLR4 mRNA gradually decreases in mother-fed but increases in formula feeding and cold asphyxia stress, correlating with induced inducible NO synthase. TLR4, inducible NO synthase, and inflammatory cytokine induction occurred in the intestinal epithelium but not in the submucosa. NEC incidence was diminished in C3H/HeJ mice, compared with C3HeB/FeJ mice. In summary, bacteria and TLR4 play significant roles in experimental NEC, likely via an interaction of intraluminal bacteria and aberrantly overexpressed TLR4 in enterocytes.

T-cell acute lymphoblastic leukemia (T-ALL), unlike other ALL types, is only infrequently associated with chromosomal aberrations, but it was recently shown that most individuals with T-ALL carry activating mutations in the NOTCH1 gene. However, the signaling pathways and target genes responsible for Notch1-induced neoplastic transformation remain undefined. We report here that constitutively active Notch1 activates the NF-kappaB pathway transcriptionally and via the IkappaB kinase (IKK) complex, thereby causing increased expression of several well characterized target genes of NF-kappaB in bone marrow hematopoietic stem cells and progenitors. Our observations demonstrate that the NF-kappaB pathway is highly active in established human T-ALL and that inhibition of the pathway can efficiently restrict tumor growth both in vitro and in vivo. These findings identify NF-kappaB as one of the major mediators of Notch1-induced transformation and suggest that the NF-kappaB pathway is a potential target of future therapies of T-ALL.

TCR-driven interactions determine the lineage choice of CD4+CD8+ thymocytes, but the molecular mechanisms that induce the lineage-determining transcription factors are unknown. Here we show that TCR-induced Egr2 and Egr1 proteins had elevated and prolonged expression in NKT lineage precursors compared with conventional lineages. ChIP-seq analysis uncovered that Egr2 directly bound and activated the promoter of Zbtb16 which encodes the NKT lineage-specific transcription factor PLZF. Egr2 also bound the Il2rb promoter and controlled the responsiveness to IL-15, which signals the terminal differentiation of the NKT lineage. Thus, we propose that elevated and persistent Egr2 levels specify the early and late stages of NKT lineage differentiation, providing a discriminating mechanism that enables TCR signaling to instruct a thymic lineage.

PLZF-expressing NKT cells establish residence at intravascular locations, failing to enter the circulation because of constitutive interactions with LFA-1 and ICAM-1.