Experimental Observations Leading to Microbial Endocrinology

The involvement of PNI in the creation of microbial endocrinology went far beyond the theoretical aspects described above. By 1992 I had obtained my first NIH grant which embodied a PNI approach examining the mechanisms by which stress could affect susceptibility to infectious disease. Although stress had been well recognized to affect susceptibility to infections for nearly 100 years (Peterson et al. 1991), I sought to identify relevant immune-based mechanisms through the use of the ethologically-relevant stress of social conflict (Fig. 1.1), instead of the more artificial stressors such as restraint stress or electric shock, which did not

Fig. 1.1 Social conflict in mice is conducted by the simple placement of a group-housed mouse also known as an "intruder" (black, C57BL/6J male) into the cage of a singly-housed mouse, also known as the "resident" (white, CF-1 male). The resident will engage the intruder ultimately resulting in the "defeat" of the intruder as shown by the limp forepaws and angled ears. Once the intruder assumes the defeat posture, the resident then disengages and at this point the intruder is removed. The social conflict procedure is done under reversed day-night light cycle using low level red light for illumination. For a fuller description of social conflict procedure see Lyte et al. (1990b) and Miczek et al. (2001)

Fig. 1.1 Social conflict in mice is conducted by the simple placement of a group-housed mouse also known as an "intruder" (black, C57BL/6J male) into the cage of a singly-housed mouse, also known as the "resident" (white, CF-1 male). The resident will engage the intruder ultimately resulting in the "defeat" of the intruder as shown by the limp forepaws and angled ears. Once the intruder assumes the defeat posture, the resident then disengages and at this point the intruder is removed. The social conflict procedure is done under reversed day-night light cycle using low level red light for illumination. For a fuller description of social conflict procedure see Lyte et al. (1990b) and Miczek et al. (2001)

reflect any sort of stress that an animal would have any evolutionary experience (Miczek et al. 2001). Among my early findings was that social conflict stress induced an increase in those immune functions, notably phagocytosis, that constitutes a first line of defense against infection (Lyte et al. 1990b). From an evolutionary perspective, the finding of increased immune responsiveness against infection made perfect sense. If an animal is wounded, then bacterial infection would almost certainly be encountered. It made little sense from the animal's perspective to have immune responsiveness decreased at a time that it was presented with an infectious challenge to its survival. What would be needed during this time of acute stress would be heightened immune activity, which was what the social conflict study had shown.

However, this surprising result presented a paradox. If immune responsiveness is increased during time of acute, ethologically-relevant stress, then why is the animal more susceptible to an infectious challenge? Most of the literature over the last century had indeed shown that stressed animals did exhibit increased susceptibility to infectious disease challenge (Peterson et al. 1991). With that in mind, I conducted a series of experiments during 1991-1992 in which social conflict stressed animals were challenged with oral pathogens such as Yersinia enteroco-litica. The results of those experiments showed the surprising result of increased mortality in stressed animals as compared to home cage controls (Fig. 1.2). Should not these animals, which showed greater than a 500% increase in phagocytic capacity

Fig. 1.2 Animals were per orally challenged with Y. enterocolitica immediately prior to social conflict stress (DEF, defeated, squares) or only handling and transport into procedure room (HC, home cage controls, circles). The stress or handling was conducted once per day for 5 days and percent survival followed for 14 days

Fig. 1.2 Animals were per orally challenged with Y. enterocolitica immediately prior to social conflict stress (DEF, defeated, squares) or only handling and transport into procedure room (HC, home cage controls, circles). The stress or handling was conducted once per day for 5 days and percent survival followed for 14 days

(Lyte et al. 1990a, b), also display increased resistance to infectious challenge and not the increased mortality (Fig. 1.2)?

It was these sets of experiments during 1991-1992 that led me to reconsider the whole concept of stress and susceptibility to infectious disease not from the perspective of the animal but from that of the infecting bacterium. For a number of reasons, the infecting organism is as highly stressed, if not more so, than the stressed host. First, most infectious agents, such as food-borne pathogens, have survived food preservation and cooking steps that result in a damaged cellular state. Upon entrance into the host, the infecting bacterium must survive the host's physical defenses such as stomach acid and then survive and proliferate within the gastrointestinal tract amid the trillions of indigenous bacteria, which rigorously maintain ecological balance among various species through means including, for example, the elaboration of bacteriocins (Riley and Wertz 2002). Central among the factors that influence the ability of any infecting microbe to survive in a host is the capacity to recognize its environment and then employ that information to initiate pathogenic processes (i.e., adherence onto epithelium) and proliferate. The central question then became, what host-derived signals would be available to an infecting bacterium that could be used to the bacterium's own advantage and ultimately survival within the host? It was at this point that I made the decision to eliminate (for the time being) the role of immunology in addressing the effect of stress on the pathogenesis of infectious disease and instead to concentrate on the role of stress on the infecting bacterium within the hostile environment of the host. In other words, were there direct effects of the host's stress response on the bacterium?

Critical to the above line of reasoning was an overlooked phenomenon of infectious disease as experienced in nature (real world) as opposed to the laboratory. That aspect specifically concerns the dose of infectious organisms that are needed to effect overt disease in the host. It is well established in food microbiology that the number of infecting organisms needed to cause food-related gastrointestinal infection can be as low as ten bacteria per gram of food (Willshaw et al. 1994). However, in the laboratory, the challenge of animals with infectious bacteria can well go as high as 1010-11 bacteria or colony forming units (CFU) per ml. Further adding to this discrepancy between real world and laboratory infectious doses is that, on average, a mouse weighs 20-25 g while a human weighs 70 kg, meaning that the dosage a laboratory animal receives is many-fold greater than what is experienced by an individual. Over the last century, a number of investigators have raised the issue of whether non-ecologically relevant doses of infectious organisms can provide complete understanding of the mechanisms that underlie the pathogenesis of infectious disease in vivo (Smith 1996). In a similar fashion, this same question can also be raised regarding in vitro studies, which utilize high (>104 CFU per ml) bacterial inoculums. Not unlike the question of how a single individual may respond to a new environment as compared to how a large group of individuals may respond to the same new environment, the survival behavior of low numbers of bacteria within the new environment of the gastrointestinal tract may radically differ from that of large numbers of bacteria. This social aspect of bacterial behavior represents the newly emerging field of sociomicrobiology (Parsek and Greenberg 2005; West et al. 2006). Specifically, the environmental signals that single or low numbers of bacteria may look for markedly differ from that sought by high numbers of bacteria. And in addition to the above point of low, not high, numbers of bacteria that contaminate food, this also applies to the vast majority of infections in general in which infecting doses of bacteria are small (<104 CFU) in number.

Thus, from the outset, one of the guiding principles in microbial endocrinology has been the use of low bacterial numbers (1-103 CFU per ml) coupled with a medium that is reflective of the in vivo milieu. Other guiding principles, such as the combination of neuroendocrine hormones and bacteria under study should be matched such that each is found to occur in the same anatomical region in vivo, have also been formulated. In addition to the chapters contained in the present book, the reader is further directed to a recent comprehensive review, which thoroughly discusses the methodological aspects of conducting microbial endocrinology-related experiments (Freestone and Lyte 2008).

The choice of the initial neuroendocrine hormones for the first experiment was based on the stress response itself and the well-known increase in catecholamines (Gruchow 1979; Woolf et al. 1992). Further, the stress-induced release of cate-cholamines had been one of the primary mechanisms that had been proposed in PNI-related research to account for the ability of stress to suppress immune responsiveness, and hence increase susceptibility to an infectious challenge (Ader and Cohen 1993; Webster Marketon and Glaser 2008). As has been recognized for many decades, the induction and sustained release of the catecholamines, especially norepi-nephrine, occurs during many forms of stress extending from psychological to surgical (Fink 2000). The Gram-negative bacterium, Y. enterocolitica, was chosen as the first bacterium to test whether a neuroendocrine hormone, namely norepinephrine, could have direct effects on growth. The results of this initial experiment in 1991, which was carried out in liquid culture using small 60 mm Petri dishes, combined a low inoculum of Y. enterocolitica (33 CFU per ml of a serum-supplemented minimal medium) with norepinephrine, epinephrine, or diluent (Fig. 1.3). In many ways, this experiment, which was the proverbial "shot in the dark," is the one that has led through many years to the creation of this current book. As shown in Fig. 1.3, there is a very small amount of visual growth evident in both the control and epinephrine supplemented plates (indicated by arrows). However, in the norepinephrine supplemented culture, there is dense growth throughout. To this day, I still remember my excitement at seeing these results. And from that day on, I effectively ceased looking at PNI-related phenomena and instead turned my research direction to the study of neuroendocrine-bacterial interactions and the creation of the field of microbial endocrinology.

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