Micronutrients at Supplemental Levels, Tight Junctions and Epithelial Barrier Function: A Narrative Review.

Disease modifiers, whether from cancer, sepsis, systemic inflammation, or microbial pathogens, all appear to induce epithelial barrier leak, with induced changes of the Tight Junctional (TJ) complex being pivotal to the process. This leak-and the ensuant breakdown of compartmentation-plays a central role in disease morbidity on many levels. Accumulation of lung water in the luminal compartment of airways was a major driver of morbidity and mortality in COVID-19 and is an excellent example of the phenomenon. Increasing awareness of the ability of micronutrients to improve basal barrier function and reduce barrier compromise in pathophysiology may prove to be a low-cost, safe, and easily administered prophylactic and/or therapeutic option amenable to large populations. The growing appreciation of the clinical utility of supplemental doses of Vitamin D in COVID-19 is but one example. This narrative review is intended to propose a general theory on how and why micronutrients-at levels above normal dietary intake-successfully remodel TJs and improve barrier function. It discusses the key difference between dietary/Recommended Daily Allowance (RDA) levels of micronutrients versus supplemental levels, and why the latter are needed in disease situations. It advances a hypothesis for why signal transduction regulation of barrier function may require these higher supplemental doses to achieve the TJ remodeling and other barrier element changes that are clinically beneficial.

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.

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 (Jm ). 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.

The multiphasic TNF-α-induced compromise of Calu-3 airway epithelial barrier function.

Purpose: Airway epithelial barrier leak and the involvement of proinflammatory cytokines play a key role in a variety of diseases. This study evaluates barrier compromise by the inflammatory mediator Tumor Necrosis Factor-α (TNF-α) in the human airway epithelial Calu-3 model. Methods: We examined the effects of TNF-α on barrier function in Calu-3 cell layers using Transepithelial Electrical Resistance (TER) and transepithelial diffusion of radiolabeled probe molecules. Western immunoblot analyses of tight junctional (TJ) proteins in detergent soluble fractions were performed. Results: TNF-α dramatically reduced TER and increased paracellular permeability of both 14C-D-mannitol and the larger 5 kDa probe, 14C-inulin. A time course of the effects shows two separate actions on barrier function. An initial compromise of barrier function occurs 2-4 hours after TNF-α exposure, followed by complete recovery of barrier function by 24 hrs. Beginning 48 hrs. post-exposure, a second more sustained barrier compromise ensues, in which leakiness persists through 144 hrs. There were no changes in TJ proteins observed at 3 hrs. post exposure, but significant increases in claudins-2, -3, -4, and -5, as well as a decrease in occludin were seen at 72 hrs. post TNF-α exposure. Both the 2-4 hr. and the 72 hr. TNF-α induced leaks are shown to be mediated by the ERK signaling pathway. Conclusion: TNF-α induced a multiphasic transepithelial leak in Calu-3 cell layers that was shown to be ERK mediated, as well as involve changes in the TJ complex. The micronutrients, retinoic acid and calcitriol, were effective at reducing this barrier compromise caused by TNF-α. The significance of these results for airway disease and for COVID-19 specifically are discussed.

Improving Transient Transfection Efficiency in a Differentiated, Polar Epithelial Cell Layer.

Polar, differentiated epithelial cell culture models (especially at confluence) are difficult to transfect compared with the higher transfection efficiencies that one obtains with relatively less differentiated, nonpolar cell culture models. Here, we sought to develop a strategy to enhance the efficiency of transfecting polar, differentiated epithelial cells. We found that chemically abrading the differentiated CACO-2 human intestinal epithelial cell layer by a trypsin and EDTA pretreatment (before the use of detergent-like transfection reagents) dramatically improved transfection efficiency in this polar, differentiated model. Although this treatment did improve the transfection efficiency, it also induced leakiness in the epithelial barrier by both opening tight junctional complexes and by creating holes in the cell layer because of low-level cell death and detachment. Thus, this approach to enhance the transfection efficiency of polar, differentiated cells will be useful for assessment of the effect of the transfected/expressed protein on (re)formation of an epithelial barrier rather than on a functional barrier itself.

Improvement of Human-Oral-Epithelial-Barrier Function and of Tight Junctions by Micronutrients.

The oral epithelium represents a major interface between an organism and its external environment. Improving this barrier at the molecular level can provide an organism added protection from microbial-based diseases. Barrier function of the Gie-3B11-human-gingival-epithelial-cell-culture model is enhanced by the micronutrients zinc, quercetin, retinoic acid, and acetyl-11-keto-ß-boswellic acid, as observed by a concentration-dependent increase in transepithelial electrical resistance and a decrease in transepithelial 14C-d-mannitol permeability. With this improvement of tight-junction (TJ)-barrier function (reduced leak) comes a pattern of micronutrient-induced changes in TJ claudin abundance that is specific to each individual micronutrient, along with changes in claudin subcellular localization. These micronutrients were effective not only when administered to both cell surfaces simultaneously but also when administered to the apical surface alone, the surface to which the micronutrients would be presented in routine clinical use. The biomedical implications of micronutrient enhancement of the oral-epithelial barrier are discussed.

The Host Microbiome Regulates and Maintains Human Health: A Primer and Perspective for Non-Microbiologists.

Humans consider themselves discrete autonomous organisms, but recent research is rapidly strengthening the appreciation that associated microorganisms make essential contributions to human health and well being. Each person is inhabited and also surrounded by his/her own signature microbial cloud. A low diversity of microorganisms is associated with a plethora of diseases, including allergy, diabetes, obesity, arthritis, inflammatory bowel diseases, and even neuropsychiatric disorders. Thus, an interaction of microorganisms with the host immune system is required for a healthy body. Exposure to microorganisms from the moment we are born and appropriate microbiome assembly during childhood are essential for establishing an active immune system necessary to prevent disease later in life. Exposure to microorganisms educates the immune system, induces adaptive immunity, and initiates memory B and T cells that are essential to combat various pathogens. The correct microbial-based education of immune cells may be critical in preventing the development of autoimmune diseases and cancer. This review provides a broad overview of the importance of the host microbiome and accumulating knowledge of how it regulates and maintains a healthy human system. Cancer Res; 77(8); 1783-812. ©2017 AACR.

Zinc reduces epithelial barrier compromise induced by human seminal plasma.

Human semen has the potential to modulate the epithelial mucosal tissues it contacts, as seminal plasma (SP) is recognized to contain both pro- and anti-barrier components, yet its effects on epithelial barrier function are largely unknown. We addressed the role of human SP when exposed to the basal-lateral epithelial surface, a situation that would occur clinically with prior mechanical or disease-related injury of the human epithelial mucosal cell layers in contact with semen. The action of SP on claudins-2, -4, -5, and -7 expression, as well as on a target epithelium whose basolateral surface has been made accessible to SP, showed upregulation of claudins-4 and -5 in CACO-2 human epithelial cell layers, despite broad variance in SP-induced modulation of transepithelial electrical resistance and mannitol permeability. Upregulation of claudin-2 by SP also exhibited such variance by SP sample. We characterize individual effects on CACO-2 barrier function of nine factors known to be present abundantly in seminal plasma (zinc, EGF, citrate, spermine, fructose, urea, TGF, histone, inflammatory cytokines) to establish that zinc, spermine and fructose had significant potential to raise CACO-2 transepithelial resistance, whereas inflammatory cytokines and EGF decreased this measure of barrier function. The role of zinc as a dominant factor in determining higher levels of transepithelial resistance and lower levels of paracellular leak were confirmed by zinc chelation and exogenous zinc addition. As expected, SP presentation to the basolateral cell surface also caused a very dramatic yet transient elevation of pErk levels. Results suggest that increased zinc content in SP can compete against the barrier-compromising effect of negative modulators in SP when SP gains access to that epithelium's basolateral surface. Prophylactic elevation of zinc in an epithelial cell layer prior to contact by SP may help to protect an epithelial barrier from invasion by SP-containing STD microbial pathogens such as HPV or HIV.

Sieving characteristics of cytokine- and peroxide-induced epithelial barrier leak: Inhibition by berberine.

AIM: To study whether the inflammatory bowel disease (IBD) colon which exhibits varying severity and cytokine levels across its mucosa create varying types of transepithelial leak. METHODS: We examined the effects of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1-ß (IL1ß) and hydrogen peroxide (H2O2) - singly and in combinations - on barrier function of CACO-2 cell layers. Our focus was on the type (not simply the magnitude) of transepithelial leak generated by these agents as measured by transepithelial electrical resistance (TER) and transepithelial flux of (14)C-D-mannitol, (3)H-Lactulose and (14)C-Polyethylene glycol as radiolabeled probe molecules. The isoquinoline alkaloid, berberine, was then examined for its ability to reduce specific types of transepithelial leak. RESULTS: Exposure to TNF-α alone (200 ng/mL; 48 h) induced a 50% decrease in TER, i.e., increased leak of Na(+) and Cl(-) - with only a marginal but statistically significant increase in transepithelial leak of (14)C-mannitol (Jm). Exposure to TNF-α + IFN-γ (200 ng/mL; 48 h) + IL1ß (50 ng/mL; 48 h) did not increase the TER change (from TNF-α alone), but there was now a 100% increase in Jm. There however was no increase in transepithelial leak of two larger probe molecules, (3)H-lactulose and (14)C-polyethylene glycol (PEG). However, exposure to TNF-α + IFN-γ + IL1ß followed by a 5 h exposure to 2 mmol/L H2O2 resulted in a 500% increase in (14)C-PEG leak as well as leak to the luminal mitogen, epidermal growth factor. CONCLUSION: This model of graded transepithelial leak is useful in evaluating therapeutic agents reducing IBD morbidity by reducing barrier leak to various luminal substances.

Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients.

The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.