Publication Database

The CLCA1 protein in innate immunity:
a mucus barrier component or signaling molecule? (2019)

Art

Hochschulschrift

Autor

Erickson, Nancy Ann (WE 12)

Quelle

Berlin: Mensch und Buch Verlag, 2019 — XII, 135 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/25168

Kontakt

Institut für Tierpathologie

Robert-von-Ostertag-Str. 15
14163 Berlin
+49 30 838 62450
pathologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

The CLCA (chloride channel regulators, calcium-activated) family comprises highlyconserved proteins with broad tissue expression patterns in many species, which have been shown to possess pleiotropic, yet not exactly determined functions, particularly in inflammatory diseases. Specifically CLCA1, which is secreted by mucus-producing cells e.g. of the respiratory and intestinal tract, is differentially expressed in diseases such as cystic fibrosis (mucoviscidosis), asthma, or chronic obstructive pulmonary disease, serving as a potential therapeutic target. Of all organ systems, CLCA1 is most highly expressed and secreted by mucus cells in the colon and has striking similarities in abundance and expression profile compared to the main structural mucin protein of the intestinal mucus barrier, Muc2. Apart from acting as a presumably structural component, CLCA1 may also function as an extracellular structuremodulating protein, i.e. as zinc-dependent metallohydrolase or as a calcium-dependent chloride channel regulator. Hence, CLCA1 as a structure-associated component of the mucus barrier is one of the most substantiated hypothetic functions, which has not been addressed to date. Here, Clca1-deficient and wild type mice were compared clinically and pathologically under unchallenged conditions as well as at early points in time of dextran sodium sulfate challenge and during fulminant colitis. Expression profiles of select mucin and Clca genes in colon tissue and the fecal microbiota composition were analyzed by reverse transcriptase quantitative polymerase chain reaction. The intestinal mucus barrier was comparatively assessed in terms of mucus penetrability, layering, and goblet cell filling by fluorescence-in situ-hybridization. In addition, the barrier integrity was determined by culture analysis of bacterial translocation into sentinel organs. Nonetheless, no differences between the genotypes were detected in naive or during dextran sodium sulfate-challenged conditions in any of the parameters analyzed. Therefore, CLCA1 does not seem to function as a structural component of the intestinal mucus barrier. Since the second most promising function of CLCA1 includes it being a signaling molecule modulating early inflammatory responses on the cytokine level, the dextran sodium sulfate challenge model was also analyzed regarding this specific effect. Despite no decisive effects on the clinical outcome, a distinct CLCA1 modulation of cytokine expression with a specific effect on chemokine (C-X-C motif) ligand 1 and interleukin 17 was observed during colitis. These findings coincide with previous respiratory challenge models, in which certain cytokines, particularly chemokine (C-X-C motif) ligand 1 and interleukin 17, were also differentially expressed between the genotypes, depending, however, on the stimuli used. This differential cytokine regulation in the dextran sodium sulfate colitis model underlines the previously hypothesized role of CLCA1 as a signaling molecule regulating cytokine expression in early innate immune response. Initial experiments regarding the human ortholog had shown a pro-inflammatory cytokine response in macrophages. Due to speciesspecific CLCA1 differences between humans and mice, caution is warranted in translatability and extrapolation of data between the species. Differences of presumable functions have been established in humans and mice, e.g. the induction of mucus cell metaplasia and the modulation of anion conductance. Therefore, murine CLCA1-mediated activation of murine alveolar and bone marrow-derived monocytes differentiated to macrophages (bone marrowderived macrophages) was analyzed. Lipopolysaccharide, the primary ligand of Toll-like receptor 4 and lipoteichoic acid, a Toll-like receptor 2 agonist, served as positive controls. Murine CLCA1 activated murine alveolar macrophages by inducing several early proinflammatory cytokines and chemokines - similar to the recently described activation of human and porcine macrophages by human CLCA1 - but failed to activate the bone marrowderived macrophages. Hence, these results may fully explain all aspects of the phenotype reported in Clca1-deficient mice infected with Staphylococcus aureus via reduced stimulation of alveolar macrophages by the lack of CLCA1, but fail to explain the contrary phenotype reported in Clca1-deficient mice during DSS colitis. Since bone marrow-derived macrophages were not activated by CLCA1 in this study, susceptibility to CLCA1 activation may be restricted to macrophages in mucosal environments where CLCA1 is normally present, such as the airways. Global gene expression analysis of CLCA1-stimulated alveolar macrophages shed new light on possible CLCA1 downstream pathways. The most strongly down-regulated gene in CLCA1-stimulated alveolar macrophages identified was the host-protective and immunomodulatory airway mucus component BPI fold containing family A member 1 (Bpifa1). BPIFA1 shares striking similarities with several functions hypothesized for CLCA1 in signaling, such as acting in immune defense mechanisms and liquid homeostasis in airway mucosal membranes, regulating cytokine gene expression in macrophages. Furthermore, BPIFA1 expression was also immunohistochemically located in mouse airway macrophages for the first time. Additionally, an in vivo Staphylococcus aureus pneumonia mouse model was analyzed regarding BPIFA1 expression, which suggests that CLCA1 may also modify BPIFA1 in airway epithelial cells. This study showed that CLCA1 may modulate early immune function not as a structural component of the mucus barrier, but as a modulator of cytokine expression in airway macrophages. The exact pathway including its putative receptor remains to be elucidated. Additionally, it should be investigated if CLCA1 may act on other effector cells than airway macrophages. The identification of BPIFA1 expression modulated by CLCA1 may be a link to explain complex downstream pathways associated with CLCA1, which remains to be addressed in the future.