Brain response to cecal infection with Campylobacter jejuni: analysis with Fos immunohistochemistry
Introduction
Infectious microorganisms and their products can, upon invasion of the host, rapidly activate the innate immune system, which in turn alters many brain-mediated functions, involved in thermoregulation, neuroendocrine control, sleep, social behavior, and cognition (Kent et al., 1992). In turn, the central nervous system, via neuroendocrine and autonomic pathways, exerts a powerful modulation on immune function, thus completing the now well-recognized interplay between the immune and peripheral and central nervous systems necessary to adequate host defense responses to invasive pathogens (reviewed in Besedovsky et al., 1996; Blalock, 1989; Maier, 2003). As bacterial pathogens are often inhaled or ingested, they can initiate an inflammatory response by the immune system’s first line defense, which subsequently alert the sensory components of the peripheral and central nervous systems. Whereas innate immune responses, such as elaboration of pro-inflammatory cytokines and prostaglandins, can alter neuronal processes in both peripheral (Gaykema et al., 1998) and central nervous systems (Elmquist et al., 1996; Ericsson et al., 1994), multiple mechanisms underlying these influences on the brain and ultimately behavior likely act in parallel (reviewed in Goehler et al., 2000; Rivest, 2001).
The majority of studies investigating brain responses to inflammatory conditions have involved acute systemic injections of bacterial cell wall products (i.e., lipopolysaccharides) by various routes of administration (e.g., intraperitoneal and intravenous injections). This experimental approach, however, may not exactly mimic neuroimmune responses elicited by naturally occurring infections, especially when these occur on the luminal side of barrier organs such as the lungs and intestines, in which pathogens adhere to the mucosal lining without entering the lymphatic system or the blood stream. As such, little is known concerning the ability of live bacterial pathogens that are encountered in daily life, e.g., by ingestion of contaminated animal food products, to evoke some form of immune response and ultimately influence brain function and behavior.
Although local infections may not evoke measurable systemic immune activation and the full spectrum of symptoms of sickness, they may lead to more insidious, subtle changes in affective states and emotional responsiveness. For example, the seeding of otherwise sterile wounds in the muscle and cranium of rats with low amounts of bacteria resulted in behavioral alterations such as open-field activity and duration of freezing (Bradfield et al., 1992). These behavioral alterations were shown to occur in the absence of any apparent immune activation. Further, during gastrointestinal infection with the common bacterial enteropathogen Campylobacter jejuni, mice developed anxiety-like behavior in the absence of overt signs of gastrointestinal illness (Lyte et al., 1998), as rodents appear tolerant to this food-borne pathogen unlike humans, in whom first-time exposure to C. jejuni often evokes significant enteric (diarrheal) illness (Wassenaar and Blaser, 1999). Taken together, findings from these studies imply that localized bacterial infections that do not seem to provoke systemic immune responses nevertheless influence brain function.
Whereas the above-mentioned studies have shown that local infections can influence behavior, these studies have not investigated the brain responses to these infections, which to enable behavior, must occur. Because challenge with a gastrointestinal infection represents an interoceptive challenge, responsive brain areas are most likely those that receive visceral sensory neural input. Whereas this prediction may seem initially counterintuitive, based on the observations that local infections seem to induce affective symptoms (i.e., anxiety) it is becoming increasingly evident that viscerosensory signals modulate affective states (Koutroumanidis et al., 2003; Miller et al., 2002; Price, 1999; Zagon, 2001).
To test the hypothesis that specific areas in the brain, likely viscerosensory but perhaps also those contributing to anxiety, respond to local gut infections, we assessed expression of the neuronal activation marker c-Fos, in the brains of mice treated either with saline or one of two doses of C. jejuni. The expression of the c-Fos protein was assessed at two different time points that correspond to time points in which behavioral alterations occur (Lyte et al., 1998). The expression of c-Fos was used as a tool for assessing activation throughout the brain to obtain a more comprehensive picture of brain activation than can be obtained by other measures of neural activity (e.g., electrophysiology). To determine whether circulating cytokines may contribute to brain activation following C. jejuni infection, serum levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were assessed using ELISA.
Section snippets
Animals
Four-week-old CF-1 male mice (n=60) were purchased from Charles River (Portage, MI), and upon receipt, maintained on a reversed day–night light cycle with lights off at 9:00 AM and on at 9:00 PM. They were housed in groups of four per cage (polypropylene with a bedding of wood shavings) with water and food available ad libitum, and were allowed to acclimate for at least 2 weeks. Forty animals were assigned into six groups, based on per oral challenge with one of the two doses of C. jejuni,
Confirmation of the Infection with C. jejuni
Microbiological analysis of cecal contents revealed persistent infection with C. jejuni in all challenged mice (data not shown). C. jejuni was not isolated from any of the control animals. Visual post-mortem analysis of each infected animal did not reveal the presence of any enlarged Peyer’s patches or lymph nodes, or other signs of inflammation. Thus, the oral administration of C. jejuni consistently resulted in colonization of the cecum, concordant with previous observations (Lyte et al., 1998
Discussion
The results from this study demonstrate that a triad of brain nuclei shows a remarkable increase in c-Fos expression in mice infected with C. jejuni as compared to uninfected mice: the nucleus of the solitary tract, the lateral parabrachial nucleus in the brainstem, and the paraventricular nucleus of the hypothalamus. These findings indicate the brain’s ability to sense and process the distant colonization of C. jejuni in the cecum, and may bear relevance to the phenomenon that this infection
Acknowledgements
We thank Chiao-Chi Chen, Nadia Badr, and Chunsheng Chen for their expert technical assistance. This study was supported by NIMH Grants MH50431 (to M.L.) and MH64648 (to L.E.G.). In addition, the authors sincerely thank the National Alliance for Research of Schizophrenia and Affective Disorders NARSAD for their generous support to Ronald Gaykema, who is the 2001 Callaghan Investigator.
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