The overarching goal of research in the Bilbo lab is to understand the mechanisms by which the immune, endocrine, and nervous systems interact, and how these interactions influence behavioral outcomes such as cognition, emotion and addiction.  The immune system is well characterized for its critical role in host defense.  Far beyond this limited role however, there is mounting evidence for the vital role the immune system plays within the brain, in both normal, “homeostatic” processes (e.g., sleep, metabolism, memory), as well as in pathology, when the dysregulation of immune molecules may occur.  We believe this recognition is especially critical in the area of brain development.  Microglia and astrocytes, the primary immunocompetent cells of the CNS, are involved in every major aspect of brain development and function, including axonal migration and synaptogenesis, synaptic pruning, apoptosis, and angiogenesis.  Cytokines such as interleukin-[IL]-1 beta, tumor necrosis factor [TNF], and IL-6 are produced by glia within the CNS, and are implicated in synaptic scaling, long-term potentiation, and neurogenesis.  Importantly, cytokines are involved in both injury and repair, and the conditions underlying these distinct outcomes are under intense investigation and debate. Notably, evidence from both animal and human studies implicates the immune system in a number of disorders with known or suspected developmental origins, including schizophrenia, autism, and cognitive dysfunction. 

    Thus, the proximate goal of our research program is to determine how seemingly disparate challenges during the perinatal period of life, such as infection, diet, stressors, or drugs of abuse, may converge on the immune system and thereby markedly influence brain development, as well as cognitive and affective behaviors throughout the remainder of the lifespan. 

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There are 4 major projects in the lab, and these are briefly outlined here:

1. Early-Life Programming of Later-Life Cognition

Bacterial infections represent the number one cause of infection in newborns worldwide, and are a significant cause and consequence of premature birth, which have increased 30% in the past 25 years. Major recent advances in maternal and perinatal medicine have greatly increased survival rates among these populations in developed countries.  However, it remains to be determined what the total impact of infection during the perinatal period may have on subsequent physiology and behavior in individuals. 

We have developed a model of postnatal bacterial infection in rats in order to explore these questions; rats are injected subcutaneously on postnatal day (P) 4 with phosphate-buffered-saline (PBS) or a non-lethal dose of live Escherichia coli.

    Our data have demonstrated that neonatal E. coli infection in pups markedly increases circulating cytokines (IL-1b, IL-6, TNF) and the primary stress hormone, corticosterone, for at least 48 hours after infection (Bilbo et al., 2005).  Within the brain, there is a significant and specific increase in IL-1b mRNA and protein, but not in other analyzed cytokines (IL-6, TNF), compared to PBS injection. These results suggest that IL-1b may be a key mediator of events occurring within the developing brain in response to infection at this time.  These data are intriguing given evidence that IL-1b levels are naturally elevated in the neonatal brain, peaking around P2, and declining thereafter into adulthood. Thus, neonatal infection leads to exaggerated IL-1b production at a time that a natural developmental peak occurs, suggesting that this may be a sensitive period for influences on this particular cytokine.

What enduring effects does such exposure have on the brain, and ultimately on behavior? 

One of the most common consequences of perinatal infection/inflammation generally is cognitive dysfunction, including learning, memory, and attention disorders. Initially, we considered two possible ways by which neonatal infection may influence memory: 1) it could directly influence the neural pathways supporting memory or, 2) it could alter how the adult animal responds to a subsequent immune challenge, thereby affecting the processes that support memory.  In the latter case, the memory impairment would only be “unmasked” by a subsequent immune challenge.  We used a model of contextual fear conditioning in order to assess memory. Rats were treated on P4 with PBS or E. coli as described previously, and were then as adults pre-exposed to a novel environment on the first day of testing.  Immediately after pre-exposure, rats from each neonatal group received no injection, saline, or a low dose immune challenge (25 mg/kg intraperitoneal bacterial LPS).  The following day rats received a single foot shock in the same environment, and were tested for memory on the third day. Only rats that experienced the combination of the infection on P4 and LPS after training displayed impaired memory for the explored context.  In contrast, rats that only experienced the P4 infection were not affected, identical to PBS controls in any condition.  Thus, the early-life infection acted as a vulnerability factor for later impairment (Bilbo et al., 2005).

  
Additional behavioral experiments have revealed the selective nature of the impairment.  The combination of P4 infection and adult LPS impaired the long-term (48 h) but not the short-term (1 h) memory for a contextual fear experience, indicating that the rats initially learned the association, but that long-term consolidation was impaired or prevented.  Moreover, this treatment had no effect on conditioned fear to a tone paired with shock.  Collectively these results support the hypothesis that the P4 infection leads to selective impairment of a long-term memory that depends on the hippocampus. 

   
Three additional points should also be mentioned: First (and most critical), an infection later in development, on P30, does not lead to LPS-induced memory impairment later in life.  These data indicate that the early-infection induced vulnerability is specifically a developmental effect, and not a general sensitizing event that can occur at any time. Second, no differences in overall activity, basal anxiety, or exploration have been observed in adulthood as a consequence of the neonatal infection.

   
In summary, the collective results yield two important conclusions.  First they support the hypothesis that the infection occurring during a sensitive period of life leads to a selective impairment in the consolidation of memories that depend on the hippocampus in adulthood. Second, the early infection produces a latent or hidden change in the immune system that is unmasked by either a second immune challenge or by preventing the effectiveness of the handling intervention. These data are well in accord with much of the human schizophrenia literature, which posits that a combination of early perinatal plus young-adult challenge (e.g., stress, infection) is required for the manifestation of the illness, the so-called “two-hit hypothesis”.

   
We have gone on to demonstrate that cytokine production is exaggerated within the hippocampus of the early-infected rats in response to adult LPS, and that preventing such expression prevents the memory impairment.  Furthermore, the exaggerated response is associated with exaggerated glial activation within cognitive regions of the brain.  Interestingly, however, cytokines are released from glia during normal learning, and are in fact necessary for learning (Williamson et al., J Neuroscience, 2011); thus, it is only exaggerated levels that are disruptive. These data provide support for an inverted “U” function for cytokines and memory, with physiological levels being important for memory, and any deviation from this range resulting in pathology and impairment.  Early-life infection appears to sensitize the “cytokine thermostat”, so to speak, with profound implications for optimal production within the brain and ultimately, cognitive function.

In summary, the continuing goal of this work is to characterize how glial-derived cytokines are important for memory formation under both normal and pathological conditions, as well as why P4 appears to be during a sensitive period for such long-term vulnerabilities in cognition to occur.

Relevant Publications:
1.  Williamson, LL, Sholar, PW, Mistry RS, Smith, SH, and Bilbo, SD.  (2011) Microglia and memory: modulation by early-life infection. Journal of Neuroscience, 31(43): 15511-21.

2.  Bilbo, SD. (2010) Early-life infection is a vulnerability factor for aging-related immune changes and cognitive decline.  Neurobiology of Learning & Memory, 94(1); 57-64
3.  Bland, ST, Beckley JT, Young S, Tsang V, Watkins LR, Maier SF, & Bilbo SD. (2010) Enduring consequences of early-life infection on glial and neural cell genesis within cognitive regions of the brain.  Brain, Behavior, & Immunity, 24, 329-338.
4.  Bilbo, SD, Biedenkapp, JC, Der-Avakian, A, Watkins, LR, Rudy, JW, & Maier, SF.  (2005) Neonatal infection-induced memory impairment following lipopolysaccharide in adulthood is prevented via caspase-1 inhibition.  Journal of Neuroscience, 25, 8000-8009.

2. Inflammation and Obesity:  Impact on cognition and affect

Maternal obesity is an escalating public health concern, and is associated with a number of adverse outcomes for both mother and baby, including gestational diabetes, stillbirth, and preeclampsia leading to preterm birth.  Beyond these acute consequences, maternal obesity may also “program” offspring for lifelong obesity and associated metabolic disorders, setting in motion a vicious cycle of propagating health problems.

Obesity is well characterized as a systemic inflammatory condition, and is also associated with cognitive disruption, suggesting a link between the two.  We have been exploring whether peripheral inflammation in maternal obesity may be transferred to the offspring brain, in particular the hippocampus, and thereby result in cognitive dysfunction.  To assess this, rat dams were fed a high saturated fat diet (SFD), a high trans fat diet (TFD), or a low fat diet (LFD) for 4 weeks prior to mating, and remained on the diet throughout pregnancy and lactation. SFD/TFD exposure significantly increased body weight in both moms and pups compared to controls.  Microglial activation markers were increased in the hippocampus of SFD/TFD pups at birth.  At weaning and in adulthood, pro-inflammatory cytokine expression was strikingly increased in the periphery and hippocampus following LPS in the SFD/TFD groups compared to controls.  Microglial activation within the hippocampus was also increased basally in SFD rats, suggesting a chronic priming of the cells.  Finally, there were marked changes in anxiety and spatial learning in SFD/TFD groups.  These effects were all observed in adulthood even after the pups were placed on standard chow at weaning, suggesting these outcomes were programmed early in life (Bilbo et al., 2010).

Some of our future directions for this work are to explore whether inflammation in the pup brain can be epigenetically transferred to subsequent generations (independent of high fat diet exposure), as well as whether the observed CNS inflammation is mediated by metabolic changes in the periphery (e.g., increased leptin, adipose tissue), or whether it involves long-term “priming” of innate CNS immune cells.

Relevant Publications:
Bilbo, SD & Tsang, V. (2010) Enduring consequences of maternal obesity for brain inflammation and behavior of offspring.  The FASEB J, 24(6); 2104-15.

3. Role of Glia in Addiction and Reward

A critical role for glial cells in addiction is becoming increasingly apparent. For instance, it has recently been demonstrated that opioids directly activate glial cells within the CNS in a nonclassical opioid receptor manner, via the innate immune system’s pattern recognition receptor, toll-like receptor (TLR) 4, and that this opioid-induced glial activation contributes strongly to their rewarding properties.  Thus, glial inhibitors such as minocycline, as well as selective TLR4 antagonism, markedly reduce opioid-induced dependence, tolerance, and reward.  These combined data are striking because we have shown that opioids such as morphine, stimulants such as cocaine, and inflammatory agents such as LPS all activate glia via the same TLR, which have been referred to as generic “danger” receptors.  Therefore, we have hypothesized that early-life exposure to drugs of abuse may have an enduring influence on an organism via its long-term activation of glia.

    We have discovered that glia within the Nucleus Accumbens (NAcc) respond to morphine with a rapid increase in cytokine/chemokine expression, which predicts future drug-induced reinstatement of morphine conditioned place preference (CPP). This glial response to morphine is markedly influenced by early-life experience - a neonatal handling paradigm that increases the quantity and quality of maternal care significantly increases baseline expression of the anti-inflammatory cytokine IL-10 within the NAcc, profoundly attenuates morphine-induced glial activation, and prevents the subsequent reinstatement of morphine CPP in adulthood. IL-10 expression within the NAcc and reinstatement of CPP are negatively correlated, suggesting a protective role for this specific cytokine against morphine-induced glial reactivity and drug-induced reinstatement of morphine CPP. Neonatal handling programs the expression of IL-10 within the NAcc early in development, and this is maintained into adulthood via decreased methylation of the IL-10 gene specifically within microglia. The effect of neonatal handling can be mimicked by pharmacological modulation of glia in adulthood with Ibudilast, which increases IL-10 expression, inhibits morphine-induced glial activation within the NAcc, and prevents reinstatement of morphine CPP. Taken together, we have identified a novel gene X early-life environment interaction on morphine-induced glial activation, and a specific role for this glial activation in drug-induced reinstatement of drug-seeking behavior.

4. Neurobiological Basis of Health Disparities:  Interactions of Maternal and Environmental Stress

The stress axis and the immune system are inextricably linked; we have been interested in the association between the stress and immune systems, and the subsequent implications for behavior for some time, and have recently become involved in a research collaboration here at Duke which is aimed at exploring these associations. The broad goal of the project is to explore how multiple environmental stressors during early development can impact neural and immune system development in children, and is an interdisciplinary collaboration between our lab, pediatrics, and investigators in the Children’s Environmental Health Initiative within the Nicholas School of the Environment here at Duke. 

Background: Although it is widely agreed that maternal and child health are influenced by multiple host, social, and environmental factors, little is known about the interactions of these forces, particularly at the neural level. Poverty in particular is associated with multiple environmental and psychological stressors, including lead exposure, poor air quality, minimum wage jobs, poor nutrition, and sub-standard housing. Important for this proposal, non-chemical stressors such as poor housing and limited social support can affect fetal physiology, maternal-offspring interaction, and postnatal physiology in ways that may increase vulnerability of the fetus or newborn to chemical stressor exposures (e.g., pollution or toxins). ‘Social’ factors have typically been treated as confounders that impair interpretation of the impact of physical agents, but are increasingly recognized as significant interacting agents that affect host vulnerability independently, likely via hormonal and immunologic responses.

At a mechanistic level, the immune system is ideally positioned to respond to both chemical (e.g., environmental pollutants) and social stressors, and in turn to communicate this information to the brain. Asthma is strongly associated with a greater Th2 than Th1 adaptive immune response, whereby high levels of interleukins (IL)-4, IL-5, and IL-13 are observed. Similarly, cognitive dysfunction and depression are each very strongly associated with a skewed pro-inflammatory (vs. anti-inflammatory) cytokine response (e.g., increased IL-1b, TNFa, and IL-6). Interestingly, many autoimmune disorders, such as Type I diabetes and colitis, are on the rise, similar to asthma, suggesting that pervasive environmental influences are changing the immune system at a population-wide level.

Our project is designed to test the hypothesis that combined stressors early in life synergize to produce long-term changes in cognitive, affective, and physiological (e.g., asthma) outcomes, via a common neuroimmune mechanism that may include shifting cytokine expression towards “pro-inflammation”.  Furthermore, seemingly disparate outcomes such as asthma and depression may exacerbate one another because they create a novel neural phenotype, involving a common CNS mechanism, which we hypothesize involves the innate immune cells of the brain and their subsequent influence on neural function in stress-responsive regions of the brain.