Nancy A. Erickson

Murine CLCA5 is uniquely expressed in distinct niches of airway epithelial cells

The murine mCLCA5 protein is a member of the chloride channel regulators, calcium-activated (CLCA) family and is suspected to play a role in airway mucus cell differentiation. Although mCLCA5 mRNA was previously found in total lung extracts, the expressing cells and functions in the naive murine respiratory tract are unknown. Therefore, mCLCA5 protein expression was identified by immunohistochemistry and confocal laser scanning microscopy using entire lung sections of naive mice. Moreover, we determined mRNA levels of functionally related genes (mClca3, mClca5, Muc5ac and Muc5b) and quantified mCLCA5-, mCLCA3- and CC10-positive cells and periodic acid-Schiff-positive mucus cells in naive, PBS-treated or Staphylococcus aureus-infected mice. We also investigated mCLCA5 protein expression in Streptococcus pneumoniae and influenza virus lung infection models. Finally, we determined species-specific differences in the expression patterns of the murine mCLCA5 and its human and porcine orthologs, hCLCA2 and pCLCA2. The mCLCA5 protein is uniquely expressed in highly select bronchial epithelial cells and submucosal glands in naive mice, consistent with anatomical locations of progenitor cell niches. Under conditions of challenge (PBS, S. aureus, S. pneumoniae, influenza virus), mRNA and protein expression strongly declined with protein recovery only in models retaining intact epithelial cells. In contrast to mice, human and porcine bronchial epithelial cells do not express their respective mCLCA5 orthologs and submucosal glands had fewer expressing cells, indicative of fundamental differences in mice versus humans and pigs.

The Goblet Cell Protein Clca1 (Alias mClca3 or Gob-5) Is Not Required for Intestinal Mucus Synthesis, Structure and Barrier Function in Naive or DSS-Challenged Mice.

The secreted, goblet cell-derived protein Clca1 (chloride channel regulator, calcium-activated-1) has been linked to diseases with mucus overproduction, including asthma and cystic fibrosis. In the intestine Clca1 is found in the mucus with an abundance and expression pattern similar to Muc2, the major structural mucus component. We hypothesized that Clca1 is required for the synthesis, structure or barrier function of intestinal mucus and therefore compared wild type and Clca1-deficient mice under naive and at various time points of DSS (dextran sodium sulfate)-challenged conditions. The mucus phenotype in Clca1-deficient compared to wild type mice was systematically characterized by assessment of the mucus protein composition using proteomics, immunofluorescence and expression analysis of selected mucin genes on mRNA level. Mucus barrier integrity was assessed in-vivo by analysis of bacterial penetration into the mucus and translocation into sentinel organs combined analysis of the fecal microbiota and ex-vivo by assessment of mucus penetrability using beads. All of these assays revealed no relevant differences between wild type and Clca1-deficient mice under steady state or DSS-challenged conditions in mouse colon. Clca1 is not required for mucus synthesis, structure and barrier function in the murine colon.

Role of goblet cell protein CLCA1 in murine DSS colitis

BackgroundThe secreted goblet cell protein CLCA1 (chloride channel regulator, calcium-activated-1) is, in addition to its established role in epithelial chloride conductance regulation, thought to act as a multifunctional signaling protein, including cellular differentiation pathways and induction of mucus production. Specifically, CLCA1 has recently been shown to modulate early immune responses by regulation of cytokines. Here, we analyze the role of CLCA1, which is highly expressed and secreted by colon goblet cells, in the course of murine dextran sodium sulfate-induced colitis.FindingsWe compared Clca1-deficient and wild type mice under unchallenged and DSS-challenged conditions at various time points, including weight loss, colon weight-length-ratio and histological characterization of inflammation and regeneration. Expression levels of relevant cytokines, trefoil factor 3 and E-cadherin were assessed via quantitative PCR and cytometric bead arrays. Lack of CLCA1 was associated with a more than two-fold increased expression of Cxcl-1- and Il-17-mRNA during DSS colitis. However, no differences were found between Clca1-deficient and wild type mice under unchallenged or DSS-challenged conditions in terms of clinical findings, disease progression, colitis outcome, epithelial defects or regeneration.ConclusionsCLCA1 is involved in the modulation of cytokine responses in the colon, albeit differently than what had been observed in the lungs. Obviously, the pathways involved depend on the type of challenge, time point or tissue environment.

Interspecies diversity of chloride channel regulators, calcium-activated 3 genes

Members of the chloride channel regulators, calcium-activated (CLCA) family, have been implicated in diverse biomedical conditions, including chronic inflammatory airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, the activation of macrophages, and the growth and metastatic spread of tumor cells. Several observations, however, could not be repeated across species boundaries and increasing evidence suggests that select CLCA genes are particularly prone to dynamic species-specific evolvements. Here, we systematically characterized structural and expressional differences of the CLCA3 gene across mammalian species, revealing a spectrum of gene duplications, e.g., in mice and cows, and of gene silencing via diverse chromosomal modifications in pigs and many primates, including humans. In contrast, expression of a canonical CLCA3 protein from a single functional gene seems to be evolutionarily retained in carnivores, rabbits, guinea pigs, and horses. As an accepted asthma model, we chose the cat to establish the tissue and cellular expression pattern of the CLCA3 protein which was primarily found in mucin-producing cells of the respiratory tract and in stratified epithelia of the esophagus. Our results suggest that, among developmental differences in other CLCA genes, the CLCA3 gene possesses a particularly high dynamic evolutionary diversity with pivotal consequences for humans and other primates that seem to lack a CLCA3 protein. Our data also help to explain previous contradictory results on CLCA3 obtained from different species and warrant caution in extrapolating data from animal models in conditions where CLCA3 may be involved.

Foreign body-induced meningoencephalitis causing seizures in a dog

A young Magyar vizsla dog was presented with seizures and progressive neurological deterioration in terms of symmetric prosencephalic signs despite induction of antiepileptic therapy. Cerebrospinal fluid (CSF) analysis and MRI findings were suggestive of an inflammatory process. This finding was confirmed at postmortem examination and a plant foreign body penetrating the cerebrum was found. Migrating plant material is an uncommon cause of inflammation in the CNS in dogs. It should be considered an aetiological differential diagnosis in dogs with inflammatory CSF and worsening neurological status.