Do We Have a Proper Model?

It has been reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) besides transcellular chloride transport, also controls the paracellular permeability of bronchial epithelium. The aim of this study was to test whether overexpressing wtCFTR solely regulates paracellular permeability of cell monolayers. To answer this question we used a CFBE41o– cell line transfected with wtCFTR or mutant F508del-CFTR and compered them with parental line and healthy 16HBE14o– cells. Transepithelial electrical resistance (TER) and paracellular fluorescein flux were measured under control and CFTR-stimulating conditions. CFTR stimulation significant decreased TER in 16HBE14o– and also in CFBE41o– cells transfected with wtCFTR. In contrast, TER increased upon stimulation in CFBE41o– cells and CFBE41o– cells transfected with F508del-CFTR. Under non-stimulated conditions, all four cell lines had similar paracellular fluorescein flux. Stimulation increased only the paracellular permeability of the 16HBE14o– cell monolayers. We observed that 16HBE14o– cells were significantly smaller and showed a different structure of cell-cell contacts than CFBE41o– and its overexpressing clones. Consequently, 16HBE14o– cells have about 80% more cell-cell contacts through which electrical current and solutes can leak. Also tight junction protein composition is different in ‘healthy’ 16HBE14o– cells compared to ‘cystic fibrosis’ CFBE41o– cells. We found that claudin-3 expression was considerably stronger in 16HBE14o– cells than in the three CFBE41o– cell clones and thus independent of the presence of functional CFTR. Together, CFBE41o– cell line transfection with wtCFTR modifies transcellular conductance, but not the paracellular permeability. We conclude that CFTR overexpression is not sufficient to fully reconstitute transport in CF bronchial epithelium. Hence, it is not recommended to use those cell lines to study CFTR-dependent epithelial transport

Paracellular transport through healthy and cystic fibrosis bronchial epithelial cell lines--do we have a proper model?

It has been reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) besides transcellular chloride transport, also controls the paracellular permeability of bronchial epithelium. The aim of this study was to test whether overexpressing wtCFTR solely regulates paracellular permeability of cell monolayers. To answer this question we used a CFBE41o- cell line transfected with wtCFTR or mutant F508del-CFTR and compered them with parental line and healthy 16HBE14o- cells. Transepithelial electrical resistance (TER) and paracellular fluorescein flux were measured under control and CFTR-stimulating conditions. CFTR stimulation significant decreased TER in 16HBE14o- and also in CFBE41o- cells transfected with wtCFTR. In contrast, TER increased upon stimulation in CFBE41o- cells and CFBE41o- cells transfected with F508del-CFTR. Under non-stimulated conditions, all four cell lines had similar paracellular fluorescein flux. Stimulation increased only the paracellular permeability of the 16HBE14o- cell monolayers. We observed that 16HBE14o- cells were significantly smaller and showed a different structure of cell-cell contacts than CFBE41o- and its overexpressing clones. Consequently, 16HBE14o- cells have about 80% more cell-cell contacts through which electrical current and solutes can leak. Also tight junction protein composition is different in 'healthy' 16HBE14o- cells compared to 'cystic fibrosis' CFBE41o- cells. We found that claudin-3 expression was considerably stronger in 16HBE14o- cells than in the three CFBE41o- cell clones and thus independent of the presence of functional CFTR. Together, CFBE41o- cell line transfection with wtCFTR modifies transcellular conductance, but not the paracellular permeability. We conclude that CFTR overexpression is not sufficient to fully reconstitute transport in CF bronchial epithelium. Hence, it is not recommended to use those cell lines to study CFTR-dependent epithelial transport

Differences in TJ length.

<p><b>A)</b> Upper image show immunostaining of ZO-1 (green) and nuclei staining (blue). Lower image shows magnifications of representative cell borders. <b>B)</b> Ratio of persistent length to contour length of all tested cell monolayers. 16HBE14o^– shows a cell-cell contact enlargement by 40% while CFBE41o^– cells and its transfected clones exhibit only a tiny enlargement by 10%. <b>C)</b> TJ lengths per area in µm per µm². 16HBE14o^– cells show 80% longer TJ lengths per area than CFBE41o^– cells. Furthermore, there is no difference in TJ lengths per area between CFBE41o^– cells and its transfected clones. Results are presented as mean ± SD (n = 7–8, p<0.05).</p

Scheme of the continuous transepithelial resistance measurement device (cTER).

<p>The ThinCert culture plate contains eight filter inserts with cell monolayers. The upper plate (lid) has six titanium electrodes for each insert, four electrodes to inject the current and two to measures the voltage. Electrodes are arranged in a way that resulted in a fairly homogenous electrical field.</p

Expression of CFTR mRNA.

<p>The relative quantity of CFTR gene expression was calculated by the 2^−ΔΔCt method, using GAPDH as the internal reference. CFTR mRNA expression value of 16HBE14o^– cells was defined as 1 and expression of all other cells were normalized to this value. CFTR mRNA levels of the other cell lines were displayed as a fold change relative to16HBE14o^–. Results are presented as mean ± SEM (n = 9–12, p<0.001).</p

Changes in transepithelial electrical resistance (cTER) upon cAMP.

<p><b>A)</b> 16HBE14o^– (triangles) and CFBE41o^– (circles) monolayer were grown on ThinCert supports. After 8–11 days in culture they obtained 800 and 500 Ω*cm² resistance respectively. After 5 min 8cpt-cAMP was added, causing dramatic TER decrease in 16HBE14o^– cells (red triangles) and an increase of TER for CFBE41o^– (green circles). Addition of the same amount of medium to control cells (open circles and triangles) did not show any effect. <b>B)</b> CFBE-WT monolayer (blue triangles) and CFBE-delF monolayer (brown circles) were grown on ThinCert supports. After 8–11 days in culture both clones obtained 500–600 Ω*cm² resistance. Stimulation with 8cpt-cAMP caused a decrease of TER in CFBE-WT cells (blue triangles) and an increase for CFBE-delF (brown circles). Addition of the same amount of medium to control cells (open circles and triangles) did not show any effect. Results are presented as mean ± SD (n = 3–6, p<0.05).</p

Fluorescein flux per TJ length.

<p><b>A)</b> Calculation of flux per TJ length revealed a significant lower value for 16HBE14o^– cells under resting conditions (red box) compared to CFBE41o^– cells (green box). Stimulation with cAMP caused a 3 fold increase for 16HBE14o^– cells (red hatched box) while CFBE41o^– cells do not respond to stimulation (green hatched box). <b>B)</b> CFBE-WT cells (blue) and CFBE-delF (brown) do not show statistically significant differences in fluorescein flux per TJ length neither under resting conditions nor upon stimulation with cAMP (hatched boxes). Data are presented as a box-plot showing raw data (circles), median (horizontal line) 25 and 75 percentile (box) and SD (whiskers (n = 7–13, p<0.05).</p

Detection of tight junction proteins.

<p>Presence of tight junction proteins known to be expressed in alveolar epithelial cells (claudin-3, -4, -5, -7) and their junctional localization in 16HBE14o^–, CFBE41o^– cells and its transfected clones was verified by Western blot (A) and confocal laser scanning microscopy (B). For densitometric evaluation of Western blots (C), all signals were normalized to β-actin. All values are expressed relative to the respective value detected in 16HBE14o^– cell layers. One-way Anova analysis revealed that claudin-3 expression differed (p<0.05) in 16HBE14o^– and CFBE41o^– clones, whereas claudin-4, -5 and -7 expression was not significantly different (n = 4). No claudin-18 expression was detected (not shown).</p

Bulletin bibliographique

Attentive depuis longtemps aux travaux de Jacques Le Brun, comme en témoignait encore le précédent « Bulletin bibliographique » (no 188), les Archives rappellent l’œuvre de ce grand maître de l’histoire de la spiritualité et des institutions chrétiennes à l’époque moderne, récemment disparu. La rubrique « L’atelier des sciences sociales du religieux » accueille trois articles consacrés au livre de Wiktor Stoczkowski, La science sociale comme vision du monde. Émile Durkheim et le mirage du salut (Gallimard, 2019). Bien relayé dans les médias culturels mais objet de nombreuses objections parmi les spécialistes, cet ouvrage est l’occasion d’un retour réflexif et critique sur la tradition durkheimienne et ses relectures. Cinq « notes critiques » entraînent le lecteur de la laïcité et la gestion de l’altérité religieuse en France, au Maghreb et au Québec, à l’hindouisme et à la « religion chinoise », en passant par les enjeux de l’autobiographie en sciences sociales des religions. Trois « lectures croisées » sont consacrées au dernier livre de Pierre Lassave, La sociologie des religions (Éditions de l’EHESS, 2019). Plus de cent recensions attestent enfin de la vitalité éditoriale des sciences sociales des religions et de leur ouverture à l’ensemble des sciences sociales. Cette livraison témoigne ainsi de la fidélité des Archives à une conviction de longue haleine : l’édition scientifique est un espace de rencontre, de controverse et de dialogue, plus précieux encore dans les temps que nous traversons. Our journal has long been attentive to the work of Jacques Le Brun, as the previous "Bulletin bibliographique" (no. 188) testified, and it recalls the work of this great master of the history of Christian spirituality and institutions in modern times, who has recently passed away. Within "The Workshop of the Social Sciences of Religion", three articles look at Wiktor Stoczkowski's book, La science sociale comme vision du monde. Émile Durkheim et le mirage du salut (Gallimard, 2019). If the book has been positively reviewed by the press, numerous objections have been raised by some specialists. Thus, this work is an opportunity for a thoughtful and critical return to the Durkheimian tradition and its rereadings. Five "critical notes" lead the reader from secularism and the management of religious otherness in France, the Maghreb and Quebec, to Hinduism and the "Chinese religion", through the issues of autobiography in the social sciences of religions. Three "cross-readings" are devoted to Pierre Lassave's latest book, La sociologie des religions (Éditions de l'EHESS, 2019). Finally, more than one hundred reviews attest to the editorial vitality of the social sciences of religions and their openness to the social sciences as a whole. This issue thus testifies to the Archives' fidelity to a long-term conviction: scientific publishing is a space for encounters, controversy, and dialogue, which is even more precious in the times we live in. Los Archives han estado atentos durante mucho tiempo a la obra de Jacques Le Brun, como atestiguaba el anterior "Bulletin bibliographique" (nº 188), y recuerdan la obra de este gran maestro de la historia de la espiritualidad y de las instituciones cristianas de los tiempos modernos, recientemente fallecido. En el "Taller de Ciencias Sociales Religiosas", tres artículos están dedicados al libro de Wiktor Stoczkowski, La science sociale comme vision du monde. Émile Durkheim et le mirage du salut (Gallimard, 2019). Bien difundida en los medios culturales pero objeto de muchas objeciones entre los especialistas, esta obra es una oportunidad para un retorno reflexivo y crítico sobre la tradición durkheimiana y sus relecturas. Cinco "notas críticas" conducen al lector desde el laicismo y la gestión de la alteridad religiosa en Francia, el Magreb y Quebec, al hinduismo y la "religión china", y a las cuestiones de la autobiografía en las ciencias sociales de las religiones. Tres "lecturas cruzadas" están dedicadas al último libro de Pierre Lassave, La sociologie des religions (Éditions de l'EHESS, 2019). Por último, más de un centenar de reseñas atestiguan la vitalidad editorial de las ciencias sociales de las religiones y su apertura al conjunto de las ciencias sociales. Este número atestigua así la fidelidad de los Archivos a una convicción a largo plazo: la publicación científica es un espacio de encuentro, de controversia y de diálogo, que es aún más precioso en los tiempos que vivimos. La nostra rivista è da tempo attenta all'opera di Jacques Le Brun, come testimonia il precedente "Bulletin bibliographique" (n. 188), e ricorda l'opera di questo grande maestro della storia della spiritualità e delle istituzioni cristiane della modernità, recentemente scomparso. La rubrica "Laboratorio delle scienze sociali delle religioni" accoglie tre articoli dedicati al libro di Wiktor Stoczkowski, La science sociale comme vision du monde. Émile Durkheim et le mirage du salut (Gallimard, 2019). Commentato positivamente dai media culturali ma oggetto di molte obiezioni da parte degli specialisti, questo lavoro è l'occasione per un ritorno riflessivo e critico sulla tradizione di Durkheimian e sulle sue riletture. Cinque "note critiche" conducono il lettore dal laicismo e dalla gestione dell'alterità religiosa in Francia, nel Maghreb e nel Québec, all'induismo e alla "religione cinese", fino ai limiti e le opportunità dell'autobiografia nelle scienze sociali delle religioni. Tre "letture incrociate" sono dedicate all'ultimo libro di Pierre Lassave, La sociologie des religions (Éditions de l'EHESS, 2019). Infine, più di cento recensioni attestano la vitalità editoriale delle scienze sociali delle religioni e la loro apertura alle scienze sociali nel loro insieme. Questo numero testimonia così la fedeltà degli Archives a una convinzione di lungo corso: l'editoria scientifica è uno spazio di incontro, di polemica e di dialogo, ancor più prezioso oggi