Sub-pyrogenic systemic inflammation impacts on brain and behavior, independent of cytokines

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Abstract

Systemic inflammation impacts on the brain and gives rise to behavioral changes, often referred to as ‘sickness behavior’. These symptoms are thought to be mainly mediated by pro-inflammatory cytokines. We have investigated the communication pathways between the immune system and brain following sub-pyrogenic inflammation. Low grade systemic inflammation was induced in mice using lipopolysaccharide (LPS); 1–100 μg/kg to mimic aspects of bacterial infection. Changes in fever, open-field activity, burrowing and consumption of glucose solution were assessed and immune activation was studied in the periphery and brain by measuring cytokine production, and immunohistochemistry to study changes in immune cell phenotype. Sub-pyrogenic inflammation resulted in changes in a species-typical, untrained behavior (burrowing) that depends on the integrity of the hippocampus. Increased expression of cytokines was observed in the periphery and selected regions of the brain which coincided with changes in behavior. However, peripheral neutralization of LPS-induced pro-inflammatory cytokines IL-1β, IL-6 and TNF-α did not abrogate the LPS-induced behavioral changes nor affect CNS cytokine synthesis. In contrast, pretreatment of mice with indomethacin completely prevented LPS-induced behavior changes, without affecting cytokine levels. Taken together, these experiments suggest a key role for prostaglandins, rather than cytokines, in communicating to the brain.

Introduction

We are continuously exposed to the risk of infection, but our immune system, which is essential for our homeostasis, prevents us from becoming ill most of the time. Inflammation is characterized by the development of specific humoral and cellular immune responses to a specific injury or a pathogen. During inflammation, a variety of soluble factors orchestrate the inflammatory response, which, under certain conditions, may lead to profound behavioral changes, including fever, malaise, lethargy and loss of appetite, impaired cognitive function and anhedonia; collectively referred to as sickness behavior (Hart, 1988, Konsman et al., 2002).

It has become apparent that there is abundant crosstalk between the peripheral immune system and the central nervous system (CNS). Numerous studies have given insight into how the brain monitors the presence of a peripheral inflammation and several mechanisms underlying the brain-mediated acute phase reaction have been described, for example following a challenge with endotoxin a fever response, and loss of appetite and activity are observed (Dantzer et al., 1999, Dantzer et al., 2000, Konsman et al., 2002). It is widely believed that the pro-inflammatory cytokines IL-1β (Bluthe et al., 2000a) IL-6 (Bluthe et al., 2000b, Cartmell et al., 2000) and TNF-α (Bluthe et al., 2000a) have a pivotal role in the onset of these symptoms. These cytokines are induced at the site of inflammation and then believed to signal to the brain by one of four mechanisms: directly across the blood–brain barrier, through sites lacking the blood–brain barrier, following peritoneal inflammation via the vagus nerve, or direct activation of brain endothelium by endotoxin (Ek et al., 1998, Konsman et al., 2000a). Once the signal induced by peripherally administered endotoxin has crossed the blood–brain barrier these same pro-inflammatory cytokines are then generated by non-neuronal cells within selected regions of the brain. Studies showing that injection of recombinant forms of the cytokines into the appropriate regions of the brain mimics these behavioral changes induced by systemic administration strengthen the idea that cytokines have a direct effect on the central nervous system (Konsman et al., 2000b, Larson and Dunn, 2001). In addition, receptors for IL-1β, TNF-α and IL-6 are expressed in many regions of the brain (Larson and Dunn, 2001, Turnbull and Rivier, 1999) and in vitro and in vivo studies suggest that signaling through these receptors occurs at picomolar concentrations of the ligand (Turnbull and Rivier, 1999).

The numerous studies on sickness behavior have been instrumental in understanding the molecular pathways of immune to brain communication. However, a number of studies have used systemic bolus injection of high doses of LPS, e.g., 500 μg/kg or more, which result in severe systemic inflammation and development of fever (Kozak et al., 1997, Sparkman et al., 2006, Wong et al., 1997). These conditions reflect symptoms of septic shock, while animals and humans typically encounter infectious pathogens that replicate in vivo and are exposed to lower concentrations of LPS over a more prolonged period of time. Recent studies showed that very low doses of LPS, well below those sufficient to cause fever or features typical of sickness behavior, impact on behavior. Administration of Salmonella equi LPS (0.8 ng/kg) to healthy young human subjects resulted in negative effects on verbal and non-verbal declarative memory functions and depression scores (Cohen et al., 2003, Reichenberg et al., 2001). Krabbe et al. reported a study in which volunteers received even lower doses of LPS (i.e., Escherichia coli, 0.06–0.2 ng/kg). This dose of LPS induced a 2- to 10-fold increase in circulating TNF-α and IL-6 and produced clear signs of inflammation as represented by a shift in concentration of leukocyte sub-populations, but without inducing a temperature change or activation of the HPA axis. In this study, performance on declarative memory tests was inversely correlated with increases in IL-6 and sTNF-R, indicating a negative effect on some areas of cognitive function and supporting a role of cytokines in brain functions independently of the HPA axis (Krabbe et al., 2005). Similarly, Wright et al. investigated a model of low level systemic/mild inflammation using typhoid vaccination, and showed that vaccination led to a significant increase in cytokines and a decline in mood, without inducing any physical symptoms or temperature changes (Wright et al., 2005). Systemic low grade inflammation is defined by a 2- to 3-fold increase in inflammatory cytokines and acute phase proteins, conditions which are also reported in aging and chronic disease (Petersen and Pedersen, 2005). Systemic inflammation may therefore have more far reaching consequences than currently recognized and very low grade inflammation, at sub-pyrogenic levels, may produce these effects.

The aim of the present study was to determine if sub-pyrogenic doses of LPS stimulate the immune system in rodents, sufficient to impact on behavior. We also sought to determine which pathways are involved in signaling to the brain at low levels of systemic challenge.

Section snippets

Mice

Adult mice (>8 weeks, Harlan, UK) were used in all experiments. They were housed in groups of 5–10 on arrival, in plastic cages with sawdust bedding, for at least a week before testing. Food and water were available ad libitum (except for the studies when glucose was substituted for water). The holding room was temperature controlled (19–23 °C) with a 12:12 h light–dark cycle (light on at 0700 h). Females were used as they can be group housed without the risk of outbreaks of aggression, and to

Behavioral response to low grade, systemic inflammation

To study if sub-pyrogenic doses of LPS impact on the brain we performed a series of behavioral tests, including a ‘burrowing’ task. It was recently discovered that mice spontaneously empty food pellets (or gravel, sand, even soiled bedding) from a tube placed in their cage. Burrowing is inhibited by lesions in the hippocampus or prefrontal cortex, and probably by damage to other, but as yet unidentified, brain regions (Deacon et al., 2002a, Deacon et al., 2002b). Baseline burrowing was similar

Discussion

Communication between systemic infection and inflammation and the brain is well known to occur and underpins the behavioral consequences of systemic infection and inflammatory diseases (sickness behavior). In previous studies, often using severe pyrogenic systemic challenges, a key role for the cytokines IL-1β, IL-6 and TNF-α has been suggested. The present study demonstrates that systemic, low grade inflammation impacts on the brain independently of peripheral cytokine production. We have

Acknowledgments

We thank UCB Celltech for the generous gift of the neutralizing cytokine antibodies and thank Marie-Rose Bluthe and Robert Dantzer for teaching us how to perform successful vagotomy. This research was supported by a grant from the BBSRC. The authors have no conflicting financial interests.

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