Date Published: March 9, 2017
Publisher: Public Library of Science
Author(s): James M. Mullin, Katherine M. Diguilio, Mary C. Valenzano, Rachael Deis, Sunil Thomas, E. Peter Zurbach, Shaheed Abdulhaqq, Luis J. Montaner, Frédéric André.
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.
With the exception of pathogens introduced during intravenous drug use, the first obstacle that a pathogen faces when invading an organism is an epithelial cell layer. For microbial pathogens as diverse as viruses, bacteria or fungi, or even parasites such as dust mites, a considerable amount of evolutionary design has gone into transiting these barriers, and ingenious mechanisms have evolved (reviewed in[1–3]). The epithelial cell layer has developed a complex array of defenses ranging from passive approaches, like mucus secretion and acid microclimates, to more active measures, such as defensins, epithelial-derived cytokines and extravasation of white blood cells—a stratified system of defense that has been excellently reviewed recently for the female reproductive tract.[4, 5] Epithelial barrier defense in the specific context of HIV has also recently been reviewed.
This current study shows that in the CACO-2 epithelial model: 1) SP at fixed dilutions can have an enhancing effect on epithelial barrier properties when presented to the basolateral surface; 2) the effect is variable across individual SP samples, with certain samples producing a significant barrier-enhancing effect, while others produce a significant barrier-compromising effect, and certain others produce no significant effect at all, an effect that is reflected in actions upon tight junctional proteins; 3) removal of zinc from SP by chelation results in increased barrier-compromising activity by SP, irrespective of sample tested; 4) re-addition of zinc to the chelated SP restores barrier function; 5) this effect is specific for zinc, and does not hold for supplementation with Ca, Mg or Cu. These data suggest that zinc is one of the components, if not the major component, in SP that minimizes or prevents SP from significantly compromising epithelial barrier function if it contacts the basolateral epithelial surface. It remains to be determined whether zinc is directly counteracting a negative modulator of barrier function or is simply one of the major positive modulators of barrier function in SP. The fact that zinc within an approximately 10–100 μM concentration window can have a positive action on barrier function makes it possible that zinc can be elevated prophylactically at the site of a “target” epithelium that will come in contact with SP, in order to minimize barrier compromise coming from SP action on this epithelium. This could be critical when the zinc content of an ejaculate is low, which could then lead to paracellular leakiness in an epithelial tissue. It could be particularly an issue when zinc is low and certain cytokines in SP, such as IL-8, are elevated. IL-8 elevation in general is known to only be involved in HIV susceptibility but also in disease progression.[25, 26] Moreover, zinc deficiency is not uncommon in the HIV-infected population.