Epithelial defence – the role of highly reactive oxygen species

Cell-cell communication is a key feature of biological systems particularly multicellular eukaryotes whose development and functioning rely on complex information exchange between and within cells and tissues and across both long and short distances. The relative roles of particular cell-cell signalling pathways is therefore of interest to a number of processes - one being the host responses during infection.

Fig.1. Typical epithelial cell layer (lung tissue in this case). Nuclei stained in blue, actin in red and tight junctions in green. Notice the physical barrier formation. http://www.seas.upenn.edu/~injury/images/091201%20triple%20slide%20test%20A5%201_5para%2040x%20overlay.jpg

The host immune systems – both innate and adaptive – require the detection of microbial pathogens and the efficient response to invasion in order to protect the host. The surfaces of our bodies are covered in epithelial cells (a thin layer of closely bound cells with characteristic surfaces which protect the underlying tissue) and these are what - along with secreted antimicrobial molecules, mucous and symbiotic bacteria - form the initial 'innate' defences against potential pathogens from our environment (fig1.). This physical barrier protects the organs below as it is generally not a good idea for these tissues to harbour replicating micro-organisms. 

The epithelium has the ability to detect and respond to the presence of pathogens in their close vicinity via a number of detector molecules and signalling pathways. The epithelial cells respond to microbial molecular 'signatures' with the production and secretion of key soluble signalling molecules facilitating inflammation (recruitment of professional immune cells to the source of infection) and antagonising microbial growth. This allows the formation of an effective immune response locally and systemically to remove the potential damage-causing source; a well-functioning early and rapid immune response is critical for protection against a number of prospective invaders.


The mystery concerning these early time-points in infections is how a small number of cells can generate an efficient immune response (and attract the required number of immune cells) despite the low number of microbes/soluble mediators.

A recent study has further investigated the ability of human epithelial cells to respond co-ordinately against these microbes – the kind found along the surfaces of our respiratory, urogenital and gastrointestinal tracts. The group used a model consisting of Listeria monocytogenes, (a facultative intracellular bacterium which can cause a number of dangerous infections in susceptible individuals and is usually acquired via the intestinal epithelium from contaminated foods) infection within an epithelial cell monolayer in vitro. The epithelial cells used were those originating from human intestines – the largest mucosal surface open to pathogenic microbes and hence could be attributable to a range of pathogen/host systems.

They were able follow how the cells responded to infection at the single cell level using GFP-expressing bacteria, flow cytometry and immunofluorescence microscopy; an approach that has significant advantages over techniques which utilise cell-culture supernatant to investigate cell-signalling and would fail to detect high-resolution single-cell changes.

Fig.2. Listeria cell entry mediated by lysis of intracellular phagosomes and further cell-cell spread facilitated by bacterial activation of actin polymerisation.

By utilising a number of L.cytogenes knock-out strains that were unable to enter the host cytoplasm and so were hence 'invisible' to the host's cytoplasmic detection system, it was evident that in order for cells to respond, they had to 'see' the infection. Surprisingly however, those cells infected were not the ones to respond the greatest and a major reaction was seen only in those uninfected cells. The group discounted any other potential mechanisms of 'indirect activation' (bacterial escape from primary host cells, extracellular activation and known signalling pathways) except the presence of an unknown intercellular communication mechanism.

They later moved on to determine the particular mechanism of epithelial communication in this system and narrowed it down to the effects of unstable, highly reactive molecules: molecules whose signalling was not disturbed using their functional knock-outs of protein secretion, gap-junction formation and ion-channel activity.

Reactive oxygen intermediates (ROIs or reactive oxygen species - ROS) are generated through normal metabolic processes and in the recent years has been seen as a key signalling molecule through diffusion from primarily infected cells to those neighbouring uninfected ones. ROIs have been established in the generation of an effective innate - and through further signalling – adaptive immune responses to infection. The group demonstrates the necessary activity of intracellular NADPH oxidases in ROI signalling in response to Listeria infection - in this case Nox4. ROI production in this manner leads to the further activation of cytoplasmic kinases which facilitated downstream immune signalling via chemokine expression in adjacent cells. However, this was only true in uninfected cells as those primarily infected are somehow inhibited in chemokine production and downstream signalling responses possibly via the high ROI concentrations or particular pathogen associated factors although this is unknown.

Fig.3. Diagram of example ROIs. http://amfpgn.org/site/wp-content/uploads/2010/08/ROS-rad..jpg

Using an L.monocytogenes versus intestinal epithelium in vitro model Dolowschiak et al. has identified a novel horizontal signalling pathway in response to bacterial infection which may facilitate appropriate innate and adaptive immune responses to potential infection. Although no in vivo models were investigated in this instance, this could easily be carried out and it would be useful to determine if epithelial-mediated ROI signalling was necessary for host protection given the complex cell-cell interactions (including the presence of key immune cells at the epithelial site of infection) in a whole organism. Also of interest would be if this would be applicable to other bacterial pathogens or indeed viral infections given the similarities between the two intracellular pathogens. The group alludes to future work examining the effect of exact ROI concentrations across epithelium and its roles in signalling.

Dolowschiak T, Chassin C, Ben Mkaddem S, Fuchs TM, Weiss S, et al. (2010) Potentiation of Epithelial Innate Host Responses by Intercellular

Communication. PLoS Pathog 6(11): e1001194. doi:10.1371/journal.ppat.1001194