Impact of Catecholamines on the Interaction of E coli with Intestinal Mucosa

As indicated earlier, increased faecal excretion of E. coli has been noted in pigs subject to social stress (Jones et al. 2001) or physical handling (Dowd et al. 2007). Evidence that such events may be correlated with the release of stress-related catecholamines is afforded by the observation that the selective neurotoxin 6-hydroxy-dopamine rapidly and dramatically increases the number of E. coli in the cecum of mice (Lyte and Bailey 1997). 6-hydroxydopamine destroys noradrenergic nerve terminals without transit across the blood-brain barrier and elicits an immediate systemic release of NE. Within 14 days, during which time noradrenergic nerves regenerate, coliform counts returned to normal (Lyte and Bailey 1997). The effect could be inhibited by prior administration of desipramine hydrochloride, which specifically inhibits catecholamine uptake into noradrenergic nerve terminals, implying that damage to such neurons is required for bacterial outgrowth (Lyte and Bailey 1997). In murine models, stress induced by partial hepatectomy or short-term starvation also caused a profound increase in the number of E. coli adhering to the cecal mucosa compared to control mice (Hendrickson et al. 1999).

In addition to evidence that release of endogenous catecholamines may alter the activities of luminal E. coli, direct instillation of NE into the gut lumen has been reported to enhance adherence of E. coli O157:H7 to the mucosal surface in a bovine ligated ileal loop model (Vlisidou et al. 2004). E. coli O157:H7 can rarely be found in association with the mucosa 12 h post-inoculation of bovine ileal loops (Fig. 6.2a), however, if combined with 5 mM NE immediately prior to inoculation,

Fig. 6.2 Confocal laser scanning micrographs of bovine mid-ileal mucosa from ligated loops inoculated with E. coli O157:H7 strain 85-170 nalR in the presence of diluent (a), or 5 mM NE (b). F-actin was stained with fluorescein isothiocyanate-conjugated phalloidin (green), and bacteria detected with rabbit anti-O157 typing serum and anti-rabbit Ig-Alexa568 (red). Dense microcolonies of intimately attached bacteria were seen only in the presence of NE. Magnification x630. Increased recruitment of neutrophils in the presence of NE was confirmed by analysis of g-emissions associated with 111In-labelled neutrophils in the same tissues as used for microscopy (Vlisidou et al. 2004). (c) Shows a transmission electron micrograph of AE lesions induced by E. coli O157:H7 in the presence of 5 mM NE. Scale bar = 5 mm. Reprinted from Vlisidou et al. (2004)

Fig. 6.2 Confocal laser scanning micrographs of bovine mid-ileal mucosa from ligated loops inoculated with E. coli O157:H7 strain 85-170 nalR in the presence of diluent (a), or 5 mM NE (b). F-actin was stained with fluorescein isothiocyanate-conjugated phalloidin (green), and bacteria detected with rabbit anti-O157 typing serum and anti-rabbit Ig-Alexa568 (red). Dense microcolonies of intimately attached bacteria were seen only in the presence of NE. Magnification x630. Increased recruitment of neutrophils in the presence of NE was confirmed by analysis of g-emissions associated with 111In-labelled neutrophils in the same tissues as used for microscopy (Vlisidou et al. 2004). (c) Shows a transmission electron micrograph of AE lesions induced by E. coli O157:H7 in the presence of 5 mM NE. Scale bar = 5 mm. Reprinted from Vlisidou et al. (2004)

dense microcolonies of adherent bacteria could be observed (Fig. 6.2b). Adherent bacteria were found to have formed "attaching and effacing" (AE) lesions that are known to be vital for bacterial persistence in the bovine intestines (Fig. 6.2c; Dziva et al. 2004; van Diemen et al. 2005). NE also significantly increased fluid accumulation and the recruitment of mIn oxinate-labelled neutrophils in response to E. coli O157:H7 compared to control loops filled with bacteria and diluent, albeit only when NE used at 5 mM and not 50 mM (Vlisidou et al. 2004). NE alone did not induce enteritis in this model, indicating that the effect was not the result of outgrowth of resident enteric bacteria or damage to intestinal mucosa.

The relevance of observations in the ligated loop model, where millimolar concentrations of NE were used, to the activities of E. coli in a stressed host is unclear. Short-term starvation prior to surgery and manipulation of the intestines to construct ligated segments does not stimulate extensive adherence of E. coli O157:H7 per se (Fig. 6.2a), and one may therefore question the relevance of the observations. However, the concentration of NE in the intestinal tract of normal and surgically manipulated calves is unknown. Quantification of tissue-associated and free NE in the intestines is difficult for several reasons (Grassi and Esler 1999; Hjemdahl 1993); (1) microdialysis probes used to sample NE would not be suitable for use in the gut owing to blockage of the dialysis membrane and the fact that NE levels in the dialysate may not reach equilibrium with the surroundings over time, (2) high-pressure liquid chromatography is required to quantify NE in gut contents and recovery through purification steps cannot be estimated, (3) breakdown products of NE can be detected in the intestines, and it is not feasible to calculate the loss of NE through the activity of host and/or bacterial enzymes or other processes. Despite these limitations, the model offers the advantage that at least ten strains or treatments can be evaluated in triplicate in the same animal relative to internal positive and negative controls, thereby minimising the impact of inter-animal variation.

Analysis of muscle-stripped intestinal explants clamped between half-tubes in Ussing chambers provides a further tractable model to probe the role of bacterial and host factors in colonization of the mucosal surface (reviewed in Chap. 5). In this system, NE promotes adherence of E. coli O157:H7 to murine cecal mucosa (Chen et al. 2003) and porcine colonic mucosa (Green et al. 2004). NE also promoted uptake of E. coli O157:H7, but not a rodent commensal E. coli strain, into porcine jejunal Peyer's Patch tissue. EHEC are not widely considered to be invasive pathogens, and uptake by such tissue may reflect the antigen-sampling activity of M cells in follicle-associated epithelium (FAE). It is therefore noteworthy that FAE derived from rats subjected to chronic psychological (water avoidance) stress was found to take up higher levels of fixed fluorophore-conjugated E. coli K-12 as compared to epithelium from control rats (Velin et al. 2004). As inactivated bacteria were used and explants were not treated with exogenous neurochemicals, the data imply that stimulation of host tissues under stress may modulate subsequent interactions with enteric bacteria. Increased bacterial uptake was not observed in villus epithelium from stressed rats, indicating that the effect may be specific to FAE.

It is important to note that NE-stimulated adherence of E. coli O157:H7 to the luminal aspect of explants clamped in Ussing chambers occurred following the application of NE to the contraluminal aspect. Though it is not possible to preclude the possibility that some NE diffused to the luminal side, the amount of diffused NE and time of exposure is considered unlikely to have been adequate to promote a substantial increase in the number of bacteria available for adherence. Measurements of short circuit current and tissue electrical conductance indicated that the mucosa was viable and intact. In addition, pre-treatment of murine cecal explants with either the non-selective a-adrenergic receptor antagonist phen-tolamine or the b-adrenergic receptor antagonist propranolol prevented the action of NE, indicating that NE-promoted bacterial adherence at least partially reflects alterations in the host tissue (Chen et al. 2003). A similar effect of phentolamine on adherence and entry of E. coli O157:H7 into porcine jejunal Peyer's patch mucosa has been reported (Green et al. 2003), however in this system, propanolol did not inhibit the effect of NE (Green et al. 2004; Chen et al. 2006). Evidence exists that the ability of NE to stimulate adherence of EHEC to porcine intestinal explants may be more sensitive to the a2-adrenergic receptor antagonist yohimbine than the a1-adrenergic antagonist prazosin (Green et al. 2004; Chen et al. 2006). The importance of release of NE from endogenous stores has also been suggested by the use of sympathomimetic drugs. For example, the NE reuptake inhibitor cocaine and a2-adrenergic receptor agonist UK-14,304 both stimulate adherence of E. coli O157:H7 to porcine colon explants (Chen et al. 2006), as does a combination of the NE reuptake blocker desipramine and pargyline, an inhibitor of monoamine oxidase that influences catecholamine metabolism (Green et al. 2004).

Though most studies with explants have focussed on NE, it has also been shown that DA increases adherence of E. coli O157:H7 to murine cecal mucosa when applied to the contraluminal aspect in a manner sensitive to the DA antagonist haloperidol (Chen et al. 2003). The stress-related peptide adrenocorticotropic hormone (ACTH) also augments adherence of E. coli O157:H7, but not a pig-adapted non-pathogenic E. coli, to porcine colonic mucosa (Schreiber and Brown 2005). Tyramine, which is structurally related to dopamine and abundant in certain dairy products owing to the tyrosine decarboxylase activity of Enterococcus faecalis starter cultures, can promote adherence of E. coli O157:H7 to murine cecal explants (Lyte 2004). In addition, extracts of banana that are rich in NE and serotonin (Waalkes et al. 1958) augment growth of Gram-negative bacteria (Lyte 1997), as do a number of non-catecholamine dietary catechols (Freestone et al. 2007c; reviewed in Chap. 4). Further studies are required to determine if neurochemicals consumed in the diet are able to modulate the outcome of enteric bacterial infections.

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