Abstract
The secreted airway mucus cell protein chloride channel regulator, calcium-activated 1, CLCA1, plays a role in inflammatory respiratory diseases via as yet unidentified pathways. For example, deficiency of CLCA1 in a mouse model of acute pneumonia resulted in reduced cytokine expression with less leukocyte recruitment and the human CLCA1 was shown to be capable of activating macrophages in vitro. Translation of experimental data between human and mouse models has proven problematic due to several CLCA species-specific differences. We therefore characterized activation of macrophages by CLCA1 in detail in solely murine ex vivo and in vitro models. Only alveolar but not bone marrow-derived macrophages freshly isolated from C57BL6/J mice increased their expression levels of several pro-inflammatory and leukotactic cytokines upon CLCA1 stimulation. Among the most strongly regulated genes, we identified the host-protective and immunomodulatory airway mucus component BPIFA1, previously unknown to be expressed by airway macrophages. Furthermore, evidence from an in vivo Staphylococcus aureus pneumonia mouse model suggests that CLCA1 may also modify BPIFA1 expression in airway epithelial cells. Our data underscore and specify the role of mouse CLCA1 in inflammatory airway disease to activate airway macrophages. In addition to its ability to upregulate cytokine expression which explains previous observations in the Clca1-deficient S. aureus pneumonia mouse model, modulation of BPIFA1 expression expands the role of CLCA1 in airway disease to involvement in more complex downstream pathways, possibly including liquid homeostasis, airway protection, and antimicrobial defense.
Highlights
Chloride channel regulator, calcium-activated 1, CLCA1, is selectively expressed by goblet and other mucin-producing cells and is secreted into the mucus layer of airways, the intestinal tract and other mucosal linings in man and mice (Gibson et al 2005; Gruber et al 1998; Leverkoehne and Gruber 2002)
Using global gene expression analyses, we further identified other genes that were differentially regulated in alveolar macrophages upon activation by CLCA1, including the host-protective and immunomodulatory airway mucus component BPI fold containing family A member 1 (Bpifa1), formerly known as short palate, lung, and nasal epithelium clone (PLUNC) 1 (SPLUNC1) protein (Britto and Cohn 2015)
Stimulation of alveolar macrophages from WT mice with CLCA1-conditioned medium (CM) resulted in upregulation of Cxcl-1, Cxcl2, Il-1β and Il-6 mRNA levels when compared to incubation with supernatant from pcDNA3.1 + vector alone (pcDNA)-transfected HEK293 cells (Fig. 2a)
Summary
Calcium-activated 1, CLCA1, is selectively expressed by goblet and other mucin-producing cells and is secreted into the mucus layer of airways, the intestinal tract and other mucosal linings in man and mice (Gibson et al 2005; Gruber et al 1998; Leverkoehne and Gruber 2002). The Clca gene is duplicated in the pig and the mouse with two or three, respectively, apparently distinctly regulated proteins, expressed in different cell types and functional niches (Patel et al 2009; Plog et al 2015). The obtained cell suspension was centrifuged at 500×g for 5 min at 4 °C and resuspended in macrophage differentiating medium containing 10 ml VLE RPMI supplemented with 1% non-essential amino acids (NEA; 100×; Biochrom AG, Berlin, Germany), 1% HEPES-Buffer (1 M; Biochrom AG), 1% sodium pyruvate (100 mM; Biochrom AG), 10% L929-CM, 10% FBS and 1% penicillin–streptomycin. Microarray analyses mRNA samples of alveolar macrophages stimulated with CLCA1-CM or incubated with pcDNA-CM as negative controls (n = 3) were subjected to mRNA gene expression profiling via microarray analysis (Hummingbird Diagnostics GmbH, Heidelberg, Germany). An adjusted p value of < 0.05 was considered significant and a log of estimated fold change (log FC) value cutoffs of 1 and − 1 were considered as limits for valid statement of lowered or elevated expressions, respectively
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