Summary Toll-like receptor (TLR) responses are regulated to avoid toxicity and achieve coordinated responses appropriate for the cell environment. We found that Notch and TLR pathways cooperated to activate canonical Notch target genes, including transcriptional repressors Hes1 and Hey1, and to increase production of canonical TLR-induced cytokines TNF, IL-6 and IL-12. Cooperation by these pathways to increase target gene expression was mediated the Notch pathway component and transcription factor RBP-J, which also contributed to lethality after endotoxin injection. TLR- and Notch-induced Hes1 and Hey1 attenuated IL-6 and IL-12 production. This Hes1- and Hey1-mediated feedback inhibitory loop was abrogated by interferon-γ (IFN-γ), which blocked TLR-induced activation of canonical Notch target genes by inhibiting Notch2 signaling and downstream transcription. These findings identify new immune functions for RBP-J, Hes and Hey proteins and provide insights into mechanisms by which Notch, TLR and IFN-γ signals are integrated to modulate specific effector functions in macrophages.

This study aimed to clarify the characteristics of rat vocal fold scarring by examining the alteration of key components in the extracellular matrix: hyaluronic acid, collagen, and fibronectin. Under monitoring with a 1.9-mm-diameter telescope, unilateral vocal fold stripping was performed, and larynges were harvested at 2, 4, 8, and 12 weeks after operation. The vocal folds were histologically analyzed with Alcian blue stain, trichrome stain, and immunofluorescence of collagen type I, collagen type III, and fibronectin. The scarred vocal folds showed less hyaluronic acid and more collagen types I and III than did the controls at all time points. Type III was stable for 12 weeks, while type I declined until 8 weeks and thereafter remained unchanged. Fibronectin increased for 4 weeks and then decreased; it was close to the control level at 8 and 12 weeks. These results suggest that the tissue remodeling process in scarred vocal folds slows down around 2 months after wounding.

These results suggest that the expression of these extracellular matrix components peaks in the period around days 3 to 5, and that the characteristics of wound healing in the vocal fold are similar to those in the skin in the early phases, but differ during the subsequent remodeling phase.

The results show a clear profile of vocal fold inflammation with corresponding changes in extracellular matrix production.

Notch-mediated lateral inhibition has been reported to regulate auditory hair cell and supporting cell development from common precursors. While the Notch effector genes Hes1, Hes5 and Hey1 are expressed in the developing cochlea, inactivation of either of them causes only mild abnormality, suggesting their functional redundancy. To explore the roles of Hes/Hey genes in cochlear development, we examined compound heterozygous or homozygous mutant mice that lacked Hes1, Hes5 and Hey1 alleles. We found that a reduction in Hes/Hey gene dosage led to graded increase of hair cell formation. However, if at least one allele of Hes1, Hes5 or Hey1 was intact, excessive hair cells were accompanied by overproduction of supporting cells, suggesting that the hair cell increase does not occur at the expense of supporting cells, and that each Hes/Hey gene functions to induce supporting cells. By contrast, when all alleles of Hes1, Hes5 and Hey1 were inactivated, the number of hair cells increased more drastically, whereas that of supporting cells was unchanged compared with control, suggesting that supporting cell formation was balanced by their overproduction and fate conversion into hair cells. The increase of the cell numbers seemed to occur after the prosensory domain formation in the mutants because the proliferation state and the size of the prosensory domain were not affected. Thus, Hes1, Hes5 and Hey1 cooperatively inhibit hair cell formation, and one allele of Hes1, Hes5 or Hey1 is sufficient for supporting cell production probably by lateral inhibition in the sensory epithelium. Strikingly, Hes/Hey mutations lead to disorganized cell alignment and polarity and to hearing loss despite hair cell overproduction. These results suggest that Hes/Hey gene dosage is essential not only for generation of appropriate numbers of hair cells and supporting cells by controlling cell proliferation and lateral inhibition but also for the hearing ability by regulating the cell alignment and polarity.