Calcitriol modifies tight junctions, improves barrier function, and reduces TNF-α-induced barrier leak in the human lung-derived epithelial cell culture model, 16HBE 14o

Abstract

Using the 16HBE 14o- human airway epithelial cell culture model, calcitriol (Vitamin D) was shown to improve barrier function by two independent metrics - increased transepithelial electrical resistance (TER) and reduced transepithelial diffusion of ¹⁴ C-D-mannitol (J_m ). Both effects were concentration dependent and active out to 168 h post-treatment. Barrier improvement associated with changes in the abundance of specific tight junctional (TJ) proteins in detergent-soluble fractions, most notably decreased claudin-2. TNF-α-induced compromise of barrier function could be attenuated by calcitriol with a concentration dependence similar to that observed for improvement of control barrier function. TNF-α-induced increases in claudin-2 were partially reversed by calcitriol. The ERK 1,2 inhibitor, U0126, itself improved 16HBE barrier function indicating MAPK pathway regulation of 16HBE barrier function. Calcitriol's action was additive to the effect of U0126 in reducing TNF- α -induced barrier compromise, suggesting that calcitriol may be acting through a non-ERK pathway in its blunting of TNF- α - induced barrier compromise. This was supported by calcitriol being without effect on pERK levels elevated by the action of TNF-α. Lack of effect of TNF- α on the death marker, caspase-3, and the inability of calcitriol to decrease the elevated LC3B II level caused by TNF-α, suggest that calcitriol's barrier improvement does not involve a cell death pathway. Calcitriol's improvement of control barrier function was not additive to barrier improvement induced by retinoic acid (Vitamin A). Calcitriol improvement and protection of airway barrier function could in part explain Vitamin D's reported clinical efficacy in COVID-19 and other airway diseases.

Keywords: ERK; Sharpe-Strumia Research Foundation; calcitriol; claudin; lung; micronutrient; tight junction; tumor necrosis factor.

FIGURE 1

Effect of 50 nM Calcitriol on 16HBE Barrier Function. (a) TER and (b) transepithelial mannitol flux rate were completed as described in Material and Methods, 48 h after treatment. Data are represented as mean ± standard error for n = 12 cell layers per condition. ***p < 0.001. (Student's t test, two‐tailed).

FIGURE 2

Concentration Dependence of Calcitriol Effects on 16HBE Barrier Function. (a) TER and (b) transepithelial mannitol flux rate were completed as described in Materials and Methods, 48 h after calcitriol treatment. n = 16 for control cell layers, n = 8 for 0.2 nM, 1 nM, 25 nM and 50 nM, n = 12 for 5 nM, n = 4 for 100 nM calcitriol ‐ treated cell layers. **p < 0.01, ***p < 0.001, One Way ANOVA, Holm‐Sidak Method.

FIGURE 3

Time Course of Calcitriol Treatment on 16HBE Barrier Function. (a) TER and (b) transepithelial mannitol flux rate were completed as described in Materials and Methods at the appropriate time point following treatment with 50 nM calcitriol. n = 4 cell layers for 17, 168 h. n = 8 cell layers for 24, 48, 96 h. *p < 0.05, **p < 0.01, ***p < 0.001 versus time matched control. (Student's t test, two‐tailed). The dotted line indicates 100% (of time‐matched control), i.e. no effect.

FIGURE 4

Effect of Calcitriol on 16HBE Tight Junctional Proteins. Confluent cell layers were treated for 48 h with 50 nM calcitriol, harvested, lysed and Western Immunoblots were performed for cytosolic (a) and detergent soluble fractions (b) as described in Material and Methods. Data are represented as mean ± standard error for n = 4 cell layers for each condition. **p < 0.01. (Student's t test, two‐tailed). Yellow bars indicate control cell layers, red bars indicate calcitriol ‐treated cell layers.

FIGURE 5

Effect of Calcitriol on TNF‐α‐Induced 16HBE Transepithelial Leak. (a) TER and (b) transepithelial mannitol flux rate were measured as described in Materials and Methods, 48 h post simultaneous treatment. Control and 150 ng/ml TNF‐α conditions had n = 6 cell layers, 50 nM calcitriol + TNF‐α had n = 14 cell layers. All other conditions had n = 8 cell layers. NS indicates no significant difference versus the TNF‐α condition. *p < 0.05, **p < 0.01 versus TNF‐α condition. (Student's t test, two‐tailed).

FIGURE 6

Effect of Preincubation with Calcitriol on TNF‐α‐Induced 16HBE Transepithelial Leak. TER was measured as described in Materials and Methods, 48 h post 150 ng/ml TNF‐α treatment. n = 4 cell layers per condition. *p < 0.05 versus TNF. NS indicates no significant difference between TNF‐α + calcitriol and TNF‐α + preincubation calcitriol. (Student's t test, two‐tailed.) “preinc” refers to 24 h preincubation with 50 nM calcitriol before the 48 h simultaneous TNF‐α + calcitriol treatment.

FIGURE 7

Effect of Calcitriol on TNF‐ α‐Induced Changes in 16HBE Claudin‐2 Levels. Confluent cell layers were treated with TNF‐α or calcitriol + TNF‐α for 48 h as described in Materials and Methods. (a) Control samples [lanes A1–A3], TNF‐ α‐treated samples [lanes B1–B3], TNF‐α + calcitriol‐treated samples [C1–C3]. (b) Optical densities of Claudin‐2 protein bands (n = 3 cell layers per condition). ***p < 0.001 versus control cell layers; ^##P < 0.01 versus TNF‐α ‐treated cell layers. (Student's t test, two‐tailed).

FIGURE 8

Effect of the ERK Inhibitor, U0126, on Dome Formation and Transepithelial Electrical Resistance in 16HBE cell layers. Phase contrast image of (a) control cell layers and (b) cell layers treated with 100 μM U0126 for 24 h. Red arrows indicate domes. Bar = 100 microns. TER (c) was measured 72 h post U0126 treatment as described in Material and Methods. n = 6 cell layers per condition. ***p < 0.001 versus control cell layers. (Student's t test, two‐tailed).

FIGURE 9

Lack of Effect of Calcitriol on 16HBE pERK Levels. Confluent cell layers were treated with control or 50 nM calcitriol‐supplemented media for 24 h prior to an additional treatment with respective media for 30 min as described in Materials and Methods. Cell layers were harvested, lysed and PAGE immunoblots were performed for phosphorylated‐ERK‐1/2 (a) and total ERK‐1/2 (c). Lane A: control; lane B: calcitriol‐treated. Two of three sample lanes are shown. Band densities of 3 separate cell samples were quantified as described in Materials and Methods (b and d). Bars represent mean ± SEM for 3 cell layers. NS = not significant vs control cell layers. (Student's t test, two‐tailed).

FIGURE 10

U0126 and Calcitriol Have Additive Effects on TNF‐α‐Induced Compromise of Transepithelial Electrical Resistance Across 16HBE Cell Layers. Cell layers were pretreated for 24 h. with 100 μM U0126 and/or 50 nM calcitriol followed by a 48‐hour treatment with 150 ng/ml TNF‐α ± U0126 and/or calcitriol. TER was then measured as described in Material and Methods. n = 6 cell layers per condition ± SEM. ***p < 0.001 versus control, ^## p < 0.01 versus TNF‐α, ^# p < 0.05 versus Vit D + TNF‐α or U0126 + TNF‐α. (Student's t test, two‐tailed).

FIGURE 11

Effect of Calcitriol and U0126 on the TNF‐α‐Induced Increase of 16HBE pERK Levels. Confluent cell layers were treated with control or 50 nM calcitriol‐supplemented media for 24 h prior to treatment with control, 150 ng/ml TNF‐α alone, TNF‐α + 50 nM calcitriol or TNF‐α + 100 μM U0126 for 30 minutes as described in Materials and Methods. Cell layers were harvested, lysed and PAGE/immunoblots were performed for phosphorylated‐ERK‐1/2 (a) and total ERK‐1/2 (c). Lane A: control; lane B: TNF‐α ‐treated; lane C: calcitriol + TNF‐α –treated; lane D: U0126 + TNF‐α ‐treated. One of three sample lanes are shown. Band densities for 3 separate immunoblots were quantified as described in Materials and Methods (b and d). Bars represent mean ± SEM for 3 cell layers. ***p < 0.001 versus control; NS (not significant) versus TNF‐α (b) or versus control (d), ^### P < 0.001 versus TNF‐α (b). (Student's t test, two‐tailed).

FIGURE 12

Effect of TNF‐ α and Calcitriol on 16HBE Caspase‐3 and LC3B Levels. Confluent cell layers were treated with calcitriol, TNF‐α, or calcitriol + TNF‐α for 48 h as described in Materials and Methods. (a) Caspase‐3 and (b) LC3B‐I and ‐II (Control samples [lanes A1–A2], TNF‐ α‐treated samples [lanes B1–B2], calcitriol‐treated samples [lanes C1–C2], TNF‐α + calcitriol‐treated samples [lanes D1–D2]). Optical densities of caspase‐3 (c) and LC3B‐II (d) protein bands (n = 3 cell layers per condition (the enclosed blots [a & b] showing 2 of 3 cell layers); ***p < 0.001 versus control cell layers; NS = not significant versus control cell layers or no significant difference between the TNF‐α and calcitriol + TNF‐α conditions. (Student's t test, two‐tailed).

FIGURE 13

Effect of Retinoic Acid and Calcitriol on 16HBE Barrier Function. TER was performed as described in Material and Methods, 48 h after treatment with 50 nM calcitriol and/or 50 μM retinoic acid. n = 20 for control and retinoic acid cell layers, n = 28 for calcitriol cell layers, n = 32 for calcitriol + retinoic acid cell layers. ***p < 0.001 versus control condition. NS indicates no significant difference for retinoic acid vs combination conditions (Student's t‐test, two‐tailed).