Investigation of the effect of some probiotic related short-chain fatty acids on respiratory epithelial barrier function

Barrier dysfunction of the mucosa and skin epithelium is involved in the pathogenesis of many diseases. The tight junction structure is the main element in establishing the epithelial barrier function. Probiotic bacteria strain and dose-dependently strengthen the barrier function of different tissue epithelium, and it is known that their products such as short-chain fatty acids (SCFA) strengthen the intestinal epithelial barrier function. However, there are limited studies on the effect of SCFAs on the barrier function of the airway epithelium. The aim of this study is to investigate the effects of some SCFAs on the respiratory epithelial barrier function. In this study, Bifidobacterium longum subsp. longum 35624 and Akkermancia muciniphila were cultured and the SCFAs they produced were determined by high pressure liquid chromatography (HPLC). The effects of commercially available quisinostat, (JNJ-26481585) and each of the SCFAs, butyrate and valerate, on intestinal epithelial barrier function were investigated by liquid-liquid interface culture method using differentiated and undifferentiated Caco-2 cells. Concentrations of 5, 10, 50, and 100 nM of quisinostat, 0.05, 0.1, 0.5, 1, 2, 4, and 8 mM of butyrate, and 0.05, 0.1, 0.5, 1, 2, and 4 mM of valerate were added to differentiated and undifferentiated Caco-2 cell cultures. Caco-2 epithelial barrier function was evaluated by transepithelial resistance (TER) measurement. Only cell culture broth was added for cell culture control. 1/105 dimethylsulfoxide was used for cytotoxicity control. The effect of valerate on airway epithelial barrier function was investigated in cultures of differentiated and undifferentiated normal human primary bronchial epithelial cell (NHBEC) and undifferentiated human asthmatic human primary bronchial epithelial cell (AIBEH). The effect of valerate on differentiated NHBEC epithelial barrier function was investigated by adding it alone or in combination with 50ng/ml IL-13 at concentrations of 0.03, 0.125, 0.5 and 2 mM from the apical compartment. In these cells, quisinostat (100nM, apical) was used as epithelial barrier function enhancer control (positive control), and epithelial barrier function disruptor IL-13 (50ng/ml, basal) was used as negative control. In addition, these two controls were added to the cell culture together and their effects were investigated. The effect of valerate on the undifferentiated NHBEC epithelial barrier function was investigated by adding it alone or together with 10ng/ml IL-13 at concentrations of 1nM, 10nM, 100nM, 1uM, 10uM and 100uM from the basal compartment. In experiments with these cells, 10nM quisinostat from the basal chamber was used as the positive control, and 10ng/ml IL-13 from the basal chamber was used as the negative control. The effect of valerate on the epithelial barrier function of undifferentiated AHBEC was investigated by administration of 10nM, 100nM, and 1uM concentrations alone from the basal compartment. In experiments with undifferentiated AHBEC culture, 10nM quisinostat from the basal chamber was used as a positive control. In experiments with primary human respiratory tract cell culture, only cell culture liquid was added for cell culture control, in which no active substance was used. In the experiments, the epithelial barrier function of the cells was evaluated by TER measurement and paracellular transition analysis. In our study, B. longum subsp. longum 35624 has been shown to produce lactate, formiate and acetate, but not propionate, butyrate or valerate. Metabolism of valerate by differentiated NHBEC cells was determined by HPLC method. In differentiated Caco-2 epithelial cells; All concentrations of valerate had no effect on TER, whereas 100nM of quinostat and 4 and 8 mM of butyrate caused a decrease in TER values. In undifferentiated Caco-2 epithelial cells; The 0.1, 0.5, 1 and 4mM concentrations of valerate and the 50 and 100nM concentrations of quinostat increased the TER values, while the 4mM concentration of butyrate caused a decrease in the TER value. In differentiated NHBEC culture, valerate did not cause any change in TER values and the amount of paracellular permeability, and it could not reverse the barrier disrupting effect of IL-13 in these cells. In the undifferentiated NHBEC culture; It was determined that valerate increased TER values compared to control, depending on the donor sample, with stimulation from the basal region, but it was shown that it could not reverse the epithelial barrier disrupting effect of IL-13 and could not strengthen the AHBEC barrier function. In our study, it was found that quisinostat and valerate strengthen the Caco-2 epithelial barrier function. It has been determined that the effect of valerate on the respiratory epithelial barrier varies depending on the donor, the differentiation status of the cells, the duration of stimulation and the way it was added to the culture (basal or apical). In addition, it has been determined that valerate is metabolized by differentiated NHBECs. It is thought that there is a need for larger studies investigating the strengthening effect of SCFAs on the respiratory system epithelial barrier function.