No abstract

Nearly 70% of cystic fibrosis (CF) patients bear the phenylalanine-508 deletion but disease severity differs greatly, and is not explained by the existence of different mutations in compound heterozygous. Studies demonstrated that genes other than CFTR relate to intestinal disease in humans and CF-mouse. Kcnn4, the gene encoding the calcium-activated potassium channel KCa3.1, important for intestinal secretion, is present in a locus linked with occurrence of intestinal CF-disease in mice and humans. We reasoned that it might be a CF-modifier gene and bred a CF-mouse with Kcnn4 silencing, finding that lethality was almost abolished. Silencing of Kcnn4 did not improve intestinal secretory functions, but rather corrected increased circulating TNF-α level and reduced intestinal mast cell increase. Given the importance of mast cells in intestinal disease additional double mutant CF-animals were tested, one lacking mast cells (C-kitW-sh/W-sh) and Stat6−/− to block IgE production. While mast cell depletion had no effect, silencing Stat6 significantly reduced lethality. Our results show that Kcnn4 is an intestinal CF modifier gene partially acting through a STAT6-dependent mechanism.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Upon demyelination, oligodendrocyte progenitor cells (OPCs) are activated and they proliferate, migrate and differentiate into myelin-producing oligodendrocytes. Besides OPCs, neural stem cells (NSCs) may respond to demyelination and generate oligodendrocytes. We have recently shown that CNS-resident pericytes (PCs) respond to demyelination, proliferate and secrete Laminin alpha2 (Lama2) that, in turn, enhances OPC differentiation. Here, we aimed to evaluate whether PCs influence the fate choice of NSCs in vitro , towards the production of new myelin-producing cells. Indeed, upon exposure to conditioned medium derived from PCs (PC-CM), the majority of NSCs gave rise to GalC- and myelin basic protein (MBP)-expressing oligodendrocytes at the expense of the generation of GFAP-positive astrocytes. Consistent with these findings, PC-CM induces an increase in the expression of the oligodendrocyte fate determinant Olig2, while the expression level of the astrocyte determinant ID2 is decreased. Finally, pre-incubation of PC-CM with an anti-Lama2 antibody prevented the generation of oligodendrocytes. Our findings indicate that PCs-derived Lama2 instructs NSCs to an oligodendrocyte fate choice favoring the generation of myelin-producing cells at the expense of astrocytes in vitro . Further studies aiming to reveal the role of PCs during remyelination may pave the way for the development of new therapies for the treatment of MS.

Calcium-activated anion secretion is expected to ameliorate cystic fibrosis, a genetic disease that carries an anion secretory defect in exocrine tissues. Human patients and animal models of the disease that present a mild intestinal phenotype have been postulated to bear a compensatory calcium-activated anion secretion in the intestine. TMEM16A is calcium-activated anion channel whose presence in the intestinal epithelium is contradictory. We aim to test the functional expression of TMEM16A using animal models with Cftr and/or Tmem16a intestinal silencing. Expression of TMEM16A was studied in a wild type and intestinal Tmem16a knockout mice by mRNA-seq, mass-spectrometry, q-PCR, Western blotting and immunolocalization. Calcium-activated anion secretion was recorded in the ileum and proximal colon of these animals including intestinal Cftr knockout and double mutants with dual Tmem16a and Cftr intestinal ablation. Mucus homeostasis was studied by immune-analysis of Mucin-2 (Muc2) and survival curves were recorded. Tmem16a transcript was found in intestine. Nevertheless, protein was barely detected in colon samples. Electrophysiological measurements demonstrated that the intestinal deletion of Tmem16a did not change calcium-activated anion secretion induced by carbachol or ATP in ileum and proximal colon. Muc2 architecture was not altered by Tmem16a silencing as was observed when Cftr was deleted from mouse intestine. Tmem16a silencing neither affected animal survival nor modified the lethality observed in the intestinal Cftr -null mouse. Our results demonstrate that TMEM16A function in the murine intestine is not related to electrogenic calcium-activated anion transport and does not affect mucus homeostasis and survival of animals.