Neonatal infection-induced memory impairment after lipopolysaccharide in adulthood is prevented via caspase-1 inhibition

“To adjust for variability in tissue sample sizes, total protein concentrations were determined by Coomassie Brilliant Blue AG-250 protein assay (Bradford, 1976).”

“Relative gene expression was determined using the 2 -⌬⌬Ct method (Livak and Schmittgen, 2001; Pfaffl, 2001).”

“Total RNA was isolated from hippocampal tissue based on the TRIzol method of Chomczynski and Sacchi (1987) and has been described in detail previously (Frank et al., 2005).”

“Relative gene expression was determined using the 2 -⌬⌬Ct method (Livak and Schmittgen, 2001; Pfaffl, 2001).”

“, 2001) and represents a time relatively comparable with the third trimester in humans during which significant brain growth occurs (Dobbing and Sands, 1979; Rodier, 1980).”

“Role of IL-1 in memory impairment Patients with acquired immunodeficiency syndrome (AIDS)related dementia, cancer, or chronic inflammatory diseases often exhibit elevated levels of IL-1 co-occurring with cognitive impairment (Gallo et al., 1989; Griffin et al., 1989; Stanley et al., 1994; Meyers, 2000).”

“IL-1 is the most consistently induced cytokine in the brain after peripheral immune stimulation and has a high density of receptors in the hippocampus (Maier and Watkins, 1998).”

“Furthermore, an increasing body of evidence from both animal (Hornig et al., 1999; Shi et al., 2003) and human (Rantakallio et al.”

“However, if a rat is preexposed to the context the day before, immediate shock conditioning will then produce substantial freezing on a subsequent test day (Fanselow, 1990; Westbrook et al., 1994; Rudy and O'Reilly, 2001).”

“Furthermore, cognitive impairment in aged or diseased populations during a subsequent infection or illness is generally more severe or prolonged compared with normal controls with the same infection/ illness (Bodles and Barger, 2004; Perry, 2004).”

“…generated by the maternal or fetal immune system during perinatal infection are important mediators of abnormal brain development and increased risk for neurodevelopmental disorders (Cai et al., 2000; Urakubo et al., 2001; Pang et al., 2003; Richardson-Burns and Tyler, 2004; Yu et al., 2004).”

“Behavioral procedures Memory was tested using a modified version of contextual fear conditioning known as the context preexposure task (Fanselow, 1990; Rudy et al., 2004).”

“Similarly, peripheral LPS increases IL-1 protein within the hippocampus (Nguyen et al., 1998) and interferes with LTP, with this effect depending on hippocampal IL-1 (Vereker et al.”

“Primer specificity (Proligo, Boulder, CO) has been verified previously in our laboratory by melt curve analysis (Johnston et al., 2004).”

“…generated by the maternal or fetal immune system during perinatal infection are important mediators of abnormal brain development and increased risk for neurodevelopmental disorders (Cai et al., 2000; Urakubo et al., 2001; Pang et al., 2003; Richardson-Burns and Tyler, 2004; Yu et al., 2004).”

“For instance, neonatal exposure to bacterial products [lipopolysaccharide (LPS)] in rats influences reactivity to stress, immune regulation, and susceptibility to disease in adulthood (Shanks et al., 2000; Hodgson et al., 2001; Boisse et al., 2004).”

“First, each stage of the procedure critically depends on the hippocampus, and the hippocampus may be particularly vulnerable to immune-related alterations (Barrientos et al., 2002; Rudy et al., 2002; Matus-Amat et al., 2004).”

“Hippocampal long-term potentiation (LTP) is thought to play a major role in the formation of many types of memory (Abraham and Williams, 2003), and exogenous IL-1 inhibits LTP within major hippocampal pathways in a dosedependent manner (Katsuki et al., 1990; Cunningham et al., 1996).”

“Total RNA was isolated from hippocampal tissue based on the TRIzol method of Chomczynski and Sacchi (1987) and has been described in detail previously (Frank et al., 2005).”

“, 2001) and represents a time relatively comparable with the third trimester in humans during which significant brain growth occurs (Dobbing and Sands, 1979; Rodier, 1980).”

“This finding is of particular interest, because memory consolidation is generally considered to occur in two stages: an early short-term memory stage and a later long-term memory stage (Igaz et al., 2002).”

“First, each stage of the procedure critically depends on the hippocampus, and the hippocampus may be particularly vulnerable to immune-related alterations (Barrientos et al., 2002; Rudy et al., 2002; Matus-Amat et al., 2004).”

“, 2003) and human (Rantakallio et al., 1997; Nelson and Willoughby, 2000) studies suggests that perinatal events involving the immune system may contribute to the development of behavioral or neuropsychiatric disorders, including schizophrenia, autism, and cerebral palsy.”

“Furthermore, an increasing body of evidence from both animal (Hornig et al., 1999; Shi et al., 2003) and human (Rantakallio et al., 1997; Nelson and Willoughby , 2000) studies suggests that perinatal events involving the immune system may contribute to the development of behavioral or…”

“Rats injected with pathological doses of IL-1 either intracerebroventricularly or directly into the dorsal hippocampus display memory impairments (Pugh et al., 1999; Barrientos et al., 2002).”

“Role of IL-1 in memory impairment Patients with acquired immunodeficiency syndrome (AIDS)related dementia, cancer, or chronic inflammatory diseases often exhibit elevated levels of IL-1 co-occurring with cognitive impairment (Gallo et al., 1989; Griffin et al., 1989; Stanley et al., 1994; Meyers, 2000).”

“For example, perinatal endotoxin administration induces cytokine expression, apoptosis, and glial activation in the immature rat brain, factors that have been linked to white matter injury (Cai et al., 2003; Golan et al., 2005).”

“…generated by the maternal or fetal immune system during perinatal infection are important mediators of abnormal brain development and increased risk for neurodevelopmental disorders (Cai et al., 2000; Urakubo et al., 2001; Pang et al., 2003; Richardson-Burns and Tyler, 2004; Yu et al., 2004).”

“Preexposure should facilitate the amount of conditioning produced by the immediate shock because it allows the rat to have established a memory of the context before the immediate shock; it is this retrieved memory representation of the context that is then associated with the immediate shock (Rudy and O’Reilly, 2001;…”

“Rats injected with pathological doses of IL-1 either intracerebroventricularly or directly into the dorsal hippocampus display memory impairments (Pugh et al., 1999; Barrientos et al., 2002).”

“Evidence from animal models suggests that cytokine actions within the brain, in particular IL-1␤, may influence cognitive processes (Banks et al., 2002).”

“Furthermore, we observed no group differences in corticosterone within the serum, a finding consistent with our previous study (Bilbo et al., 2005) but in contrast to several reports that neonatal LPS exposure potentiates hypothalamic-pituitary-adrenal (HPA) activity in adult rodents (Shanks et al.”

“There is also evidence that IL-1 contributes to demyelination and blood– brain barrier breakdown, processes that can disrupt communication among brain regions (Ferrari et al., 2004).”

“YVAD-cmk administration induces neuroprotection after cerebral ischemia, an effect that is attributed to decreases in both apoptotic mechanisms and IL-1 production (Rabuffetti et al., 2000).”

“, 2003) and human (Rantakallio et al., 1997; Nelson and Willoughby, 2000) studies suggests that perinatal events involving the immune system may contribute to the development of behavioral or neuropsychiatric disorders, including schizophrenia, autism, and cerebral palsy.”

“, 1998) and interferes with LTP, with this effect depending on hippocampal IL-1 (Vereker et al., 2000).”

“Hippocampal long-term potentiation (LTP) is thought to play a major role in the formation of many types of memory (Abraham and Williams, 2003), and exogenous IL-1 inhibits LTP within major hippocampal pathways in a dosedependent manner (Katsuki et al.”

“For example, perinatal endotoxin administration induces cytokine expression, apoptosis, and glial activation in the immature rat brain, factors that have been linked to white matter injury (Cai et al., 2003; Golan et al., 2005).”

“Primer sequences used for amplification of rat cDNAs via the ICM have demonstrated a pattern of labeling throughout the brain within 1.5 h (Proescholdt et al., 2000).”

“Role of IL-1 in memory impairment Patients with acquired immunodeficiency syndrome (AIDS)related dementia, cancer, or chronic inflammatory diseases often exhibit elevated levels of IL-1 co-occurring with cognitive impairment (Gallo et al., 1989; Griffin et al., 1989; Stanley et al., 1994; Meyers, 2000).”

“Microglia and astrocytes are resident immunocompetent cells of the CNS and, in addition to providing support to neurons, play a vital role in the brain response to infection as a major source of cytokines (Benveniste, 1997; Streit, 2002).”

“For this reason, the hippocampus is thought to be particularly vulnerable to immune-related alterations (Lynch et al., 2004).”

“For instance, neonatal exposure to bacterial products [lipopolysaccharide (LPS)] in rats influences reactivity to stress, immune regulation, and susceptibility to disease in adulthood (Shanks et al., 2000; Hodgson et al., 2001; Boisse et al., 2004).”

“Furthermore, cognitive impairment in aged or diseased populations during a subsequent infection or illness is generally more severe or prolonged compared with normal controls with the same infection/ illness (Bodles and Barger, 2004; Perry, 2004).”

“, Alzheimer’s disease and AIDS-related dementia), and astrocyte reactivity in aged rats is positively correlated with cognitive impairment (Sugaya et al., 1996).”

“, 2001; Hodgson and Knott, 2002), and yet others report no change (Granger et al., 1996; Breivik et al., 2002), consistent with our own findings.”

“For instance, neonatal exposure to bacterial products [lipopolysaccharide (LPS)] in rats influences reactivity to stress, immune regulation, and susceptibility to disease in adulthood (Shanks et al., 2000; Hodgson et al., 2001; Boisse et al., 2004).”

“Early exposure to infectious agents may have significant consequences for the development and function of physiological systems throughout an individual's lifespan, a phenomenon termed " perinatal programming " (Barker et al., 1995).”

“YVAD-cmk reduces death from sepsis after a lethal dose of LPS, independent of serum reductions in IL-1 (Mathiak et al., 2000).”

“, 2001; Hodgson and Knott, 2002), and yet others report no change (Granger et al., 1996; Breivik et al., 2002), consistent with our own findings.”

“…generated by the maternal or fetal immune system during perinatal infection are important mediators of abnormal brain development and increased risk for neurodevelopmental disorders (Cai et al., 2000; Urakubo et al., 2001; Pang et al., 2003; Richardson-Burns and Tyler, 2004; Yu et al., 2004).”

“…generated by the maternal or fetal immune system during perinatal infection are important mediators of abnormal brain development and increased risk for neurodevelopmental disorders (Cai et al., 2000; Urakubo et al., 2001; Pang et al., 2003; Richardson-Burns and Tyler, 2004; Yu et al., 2004).”

“, 2002), some report altered stress-induced activity only (Hodgson et al., 2001; Hodgson and Knott, 2002), and yet others report no change (Granger et al.”

“YVAD-cmk also prevents peroxynitrite-induced neurotoxicity in neuronal cultures, a metabolite of nitric oxide and strong oxidizing agent thought to be involved in sepsis, inflammation, and apoptosis (Zhang and Rosenberg, 2004).”

“Specifically, peripheral infection with Escherichia coli on postnatal day 4 in rats resulted in markedly impaired learning and memory due to an inflammatory (LPS) challenge in adulthood (Bilbo et al., 2008, 2005a,b, 2007).”

“coli challenge showed no differences in TNF-α concentration as compared to the control group, independent of whether or not microglia were activated [37].”

“In contrast, expression levels of the astrocyte cell marker glial-fi brillary acidic protein (GFAP) did not change, suggesting a selective change in microglial activation after neonatal infection (Bilbo et al., 2005a).”

“Since P4 rats respond to peripheral infection with robust CNS inflammation (Bilbo et al., 2005), this age-dependent seizure-induced inflammation may be ascribed to uncoupling between seizures and activation of transcriptional factors promoting inflammation such as AP-1 or NFkB (Rizzi et al., 2003).”

“Results demonstrated that only rats that experienced the combination of neonatal infection and subsequent LPS exposure displayed impaired memory for the explored context [35].”

“However, the deficit is only observed if the rats infected as newborns receive a systemic injection of LPS around the time of learning (Bilbo et al., 2005a; Bilbo et al., 2005b).”

“Furthermore, this neuro-inflammation-induced reduction in BDNF resulted in hippocampal-dependent memory deficits (Barrientos et al., 2004, 2006; Bilbo et al., 2005; Barrientos, 2011).”

“The author receives support from the European Union's Seventh Framework Programme (FP7/2007-2011) under grant agreement no. 259679.”

“However, time points, chosen to correlate findings with our read-outs on cerebral apoptosis and gliosis might have been too late and acute treatment effects on cytokine responses might have resolved (Bilbo et al., 2005; Claypoole et al., 2016; Kendall et al., 2011).”

“body, may be prolonged in the CNS for an extended period (Shi et al., 2003; Bilbo et al., 2005; Galic et al., 2008).”

“Blocking IL-1β release in the CNS prevents the memory impairment caused by overactivation of microglia (99).”

“While there were no deficits in memory function in the young adult, deficits occurred upon a secondary challenge with LPS (Bilbo et al., 2005) or with aging to 16 months (Bilbo, 2010).”

“coli-infected mice has been shown to be primed, and displays an exacerbated response to a ‘second hit’ with a low-dose of LPS, in adulthood (Bilbo et al., 2005; Williamson et al., 2011).”

“There has been increasing interest in the role of proinflammatory cytokines in modulating learning and memory processes (Barrientos et al., 2006a; Barrientos et al., 2002; Bilbo et al., 2005; Cunningham et al., 1996; Godbout et al., 2005; Hauss-Wegrzyniak et al., 1998; Pugh et al., 1998; Yirmiya et al., 2002).”

“This exciting concept describes transcriptional regulation in individual cells by probabilistic events and could explain heterogeneous expression among a cell population as occurring by chance.”

“Moreover, animal studies of MIA consistently report alteration in the ACC and insula, as well as altered inhibition, emotional regulation, and attention to salience features (Patterson, 2002; Bilbo et al., 2005; Patterson, 2009; Bland et al., 2010; Short et al., 2010; Malkova et al., 2012).”

“In neonatal rats, the effect of endotoxin on memory was attenuated by ICE blockade (Bilbo et al., 2005), indicating that IL-1 mediates this.”

“This may be important since elevated CNS pro-inflammatory cytokines, including IL-1β and/or TNFα, have been associated with increased anxiety, decreased cognition, and altered social behaviors [35-38,62].”

“Specifically, rats treated with E. coli as neonates that are given a caspase-1 inhibitor, which prevents the synthesis of the proinflammatory cytokine IL-1, do not show LPS-induced memory impairment (Bilbo et al., 2005b).”

“Currently, less is known about the long-term effect of early life infection on cytokine content in the brain, although Bilbo et al. (2005) have observed that rats injected neonatally (P4) with E. coli show exaggerated IL-1 responses to LPS in the hippocampus and cortex at adulthood.”

“Early life E. coli infections in rats have also been shown to prime the brain to a second immune challenge with LPS in adulthood (Bilbo et al., 2005).”

“However, these impairments are only observed if a second immune challenge (bacterial lipopolysaccharide [LPS]) is administered in close proximity to learning in adulthood (Bilbo et al., 2005a,b).”

“Additionally, it has been shown that neonatal immune challenges impact memory and cognitive ability in adults [21-24].”

“Consistent with this possibility, neonatally-infected rats exhibit prolonged IL-1β responses to LPS within the brain compared to control rats, and preventing the synthesis of IL-1β completely prevents the memory impairment (Bilbo et al. 2005a).”

“In addition, prenatal and neonatal LPS exposure causes long-term memory impairments (Hao et al., 2010; Bilbo et al., 2005).”

“production with a caspase I inhibitor [42,43].”

“These data suggest that IL-1b participates in the set of immune/inflammatory events resulting from neonatal infection and which likely contribute to cognitive alterations [151].”

“There were no significant differences in glial markers in any brain region of the 2 months rats, which is largely consistent with previous findings, in which functional differences emerge only after a challenge in young adults (Bilbo et al., 2005a; Bland et al., 2010).”

“This hypothesis is consistent with our finding that neonatally infected rats exhibit exaggerated IL-1 responses to LPS within the hippocampus compared to control rats, and that this altered response is an important mediator of the memory impairment [ 7 ].”

“We have demonstrated that rats infected with bacteria as neonates exhibit profound long-term memory impairments in adulthood, but only if a peripheral immune challenge (lipopolysaccharide; LPS) is given around the time of learning (Bilbo et al., 2005a,b, 2006).”

“This increase persisted into adulthood and was exaggerated by a second challenge with LPS [108,109].”

“In line with this view, it has been shown that LPS administration impairs contextual fear conditioning [8-10] and spatial memory [2,11-14] and avoidance learning [15-19] in rats and mice.”

“Thus, the hippocampus is thought to be particularly vulnerable to immune-related alterations (Bilbo et al., 2005).”

“Early-life infection generally leads to a direct elevation of circulating glucocorticoids in early-life (Bilbo et al., 2005a) and while basal CORT was not affect by early-life infection at P14, the level of phosphorylated GR is significantly higher in the prefrontal cortex, but not the hippocampus…”

“This impairment, however, is only observed if a peripheral immune challenge (lipopolysaccharide [LPS]) is administered either 24 h before or immediately after learning (Bilbo et al., 2005a,b, 2006).”

“Notably, neonatal rats infected with Escherichia coli exhibit cognitive deficits after a second immune challenge by LPS in adulthood (Bilbo et al., 2005a; Williamson et al., 2011), and P7-P14 offspring of dams injected during gestation with poly I:C (a TLR3 agonist that mimics viral infections), or…”

“Neonatal infection attenuated corticosterone response to an acute stressor, impaired cognition, and decreased social interactions in rats (Bilbo et al., 2005; Bilbo and Schwarz, 2012). http://dx.doi.org/10.1016/j.bbi.2014.02.008 0889-1591/ 2014 Elsevier Inc.”

“Inflammatory insults with live bacteria on postnatal day (P) 4 have been associated with altered fear memory (Bilbo et al., 2005) and motor coordination (Lieblein-Boff et al., 2013), as well as potentiation of glial and cytokine responses in the adult rat (reviewed in Bilbo and Schwarz, 2012),…”

“This observation is further supported by animal studies, where neonatal bacterial infection of rats induces hippocampus-dependent memory deficits in adulthood [77].”

“These data confirm previous analyses of cytokines and chemokines within the brain [7], and complement data implicating IL-1β as a critical molecule underlying the adult cognitive impairments [6,7].”

“Additionally, it has been shown that neonatal immune challenges alter memory, cognitive ability and pain sensitivity as adults (Gilmore et al. 2003, 2005; Zuckerman & Weiner, 2005; Bilbo et al. 2005; Boisse et al. 2005).”

“, 2003), as well as host defence responses, such as fever and sickness behaviour (Takahashi et al., 1996; Dantzer et al., 1998; Schneider et al., 1998; Rachal et al., 2001; Watkins et al., 2001; Ross et al., 2003; Bilbo et al., 2005).”

“…stage relatively comparable to the third trimester in humans during which significant brain growth occurs (Dobbing and Sands, 1979; Rodier, 1980), increases both peripheral and brain cytokines, as well as corticosterone for several hours (>48) following the infection (Bilbo et al., 2005a).”

“In contrast, low-dose, neonatal LPS alters future immune responses and causes memory impairment in adults (Bilbo et al., 2005).”

“In addition, in animals exposed to inflammation in utero or during early postnatal life, long-term behavioural alterations such as deficits in prepulse inhibition test [17, 18], motor behaviour [19], and learning and memory [19, 20] have also been reported.”

“Such perinatal immune activation has been associated not only with change in behavior in neonatal animals but also disrupted avoidance learning in male, but not female subjects in adulthood (69).”

“These effects include an enhanced febrile response, elevated central and peripheral cytokine activity, and increased central COX-2 levels and glia activity, and appear in large part to reflect reduced tolerance to later immunological challenges (Bilbo et al., 2005a,2007a,2008a).”

“Some studies have linked poor cognitive performance following neonatal challenge with LPS (Harré et al., 2008) or IL-1 (Favrais et al., 2011), whereas other studies have not (Bilbo et al., 2005a,b, 2006).”

“It is also known that the HP is vulnerable to immune-related alterations and important to the pathology of behavioral and cognitive disorders (Lynch et al., 2004; Bilbo et al., 2005).”

“impairment after a subsequent immune challenge later in life [1].”

“The results of present study confirmed LPS-induced learning and memory impairments which had been repeatedly reported (63-65).”

“Diseases such as SZ have also been linked to infection, fetal malnutrition, or hypoxia in early life [107-110], thus suggesting a role for environmental factors acting in the pre–conceptual, fetal, and infantile phases of life [111].”

“As with IL-6 production, LPS is more potent than poly I:C at inducing an increase in the concentration of IL-1b in the circulation (Fortier et al. 2004; Bilbo et al. 2005).”