Date Published: June 6, 2019
Publisher: Public Library of Science
Author(s): Andrea Méndez, Diego A. Rojas, Carolina A. Ponce, Rebeca Bustamante, Caroll J. Beltrán, Jorge Toledo, Victor A. García-Angulo, Mauricio Henriquez, Sergio L. Vargas, Y. Peter Di.
Clara cells are the main airway secretory cells able to regenerate epithelium in the distal airways through transdifferentiating into goblet cells, a process under negative regulation of the Notch pathway. Pneumocystis is a highly prevalent fungus in humans occurring between 2 and 5 months of age, a period when airways are still developing and respiratory morbidity typically increases. Pneumocystis induces mucus hyperproduction in immunocompetent host airways and whether it can stimulate Clara cells is unknown. Markers of Clara cell secretion and Notch1 activation were investigated in lungs of immunocompetent rats at 40, 60, and 80 days of age during Pneumocystis primary infection with and without Valproic acid (VPA), a Notch inducer. The proportion of rats expressing mucin increased in Pneumocystis-infected rats respect to controls at 60 and 80 days of age. Frequency of distal airways Clara cells was maintained while mRNA levels for the mucin-encoding genes Muc5B and Muc5ac in lung homogenates increased 1.9 and 3.9 times at 60 days of infection (P. = 0.1609 and P. = 0.0001, respectively) and protein levels of the Clara cell marker CC10 decreased in the Pneumocystis-infected rats at 60 and 80 days of age (P. = 0.0118 & P. = 0.0388). CC10 and Muc5b co-localized in distal airway epithelium of Pneumocystis-infected rats at day 60. Co-localization of Muc5b and Ki67 as marker of mitosis in distal airways was not observed suggesting that Muc5b production by Clara cells was independent of mitosis. Notch levels remained similar and no transnucleation of activated Notch associated to Pneumocystis infection was detected. Unexpectedly, mucus was greatly increased at day 80 in Pneumocystis-infected rats receiving VPA suggesting that a Notch-independent mechanism was triggered. Overall, data suggests a Clara to goblet cell transdifferentiation mechanism induced by Pneumocystis and independent of Notch.
Clara or Club cells are a group of epithelial cells in the airway which secrete Clara Cell Secretory Protein (CCSP or CC10). They are the most abundant cells in the airway of rodents (57%) and their proportion may vary among different species. In humans, Clara cells represent 22% of epithelial cells in distal airway, the location where they are more abundant. Clara cells have functions in immune response, metabolism of toxic substances and epithelial regeneration[4–6]. Moreover, these cells are considered the major Transit Amplifying (TA) cell population in the airway epithelium, which regenerate epithelial cells in normal lungs as has been documented after a lung injury in mice.
The first part of this work documented an increase in the proportion of rats with epithelial mucins in distal airway observed in the day 60 and 80 of infection, before the point of highest burden of Pneumocystis occurring after 80 days in a previous model of primary Pneumocystis infection . The present results are similar to previous studies in showing increase in epithelial mucins in the day 60 of infection by Pneumocystis. Of note, distinct niches of murine airway epithelial cells may express different mucus markers  and may also explain differences in mucin expression between rat studies that study different sections of the lung . This study focuses in distal airways with diameter 50–250 μm and previous studies showed mucins or mucus in total airways [25, 26, 28]. Pathological changes in distal airways are relevant to study because obstruction of this region affects the distribution of ventilation and may lead to small airway closure . Methacholine sensitivity has been explored in immunocompetent mice sensitized with OVA and infected with Pneumocystis via the intratracheal route and document that this fungus impressively increases airway sensitivity to methacholine [25, 39]. This increase is comparable to the effect of the common allergen House Dust Mite . Whether this effect occurs in rats spontaneously infected with Pneumocystis or other mammals in addition to mice has not been determined. The level of epithelial mucins observed in this study was lower to that of previous studies by our group , and this difference can be attributed to variations in the timing of greater mucin induction. This timing may vary between models of primary infection that use the aerial route of natural contagion without a fixed inoculum as occurs with Pneumocystis infection in infants.