Robert H. McCusker

Immune–neural connections: how the immune system’s response to infectious agents influences behavior

Summary Humans and animals use the classical five senses of sight, sound, touch, smell and taste to monitor their environment. The very survival of feral animals depends on these sensory perception systems, which is a central theme in scholarly research on comparative aspects of anatomy and physiology. But how do all of us sense and respond to an infection? We cannot see, hear, feel, smell or taste bacterial and viral pathogens, but humans and animals alike are fully aware of symptoms of sickness that are caused by these microbes. Pain, fatigue, altered sleep pattern, anorexia and fever are common symptoms in both sick animals and humans. Many of these physiological changes represent adaptive responses that are considered to promote animal survival, and this constellation of events results in sickness behavior. Infectious agents display a variety of pathogen-associated molecular patterns (PAMPs) that are recognized by pattern recognition receptors (PRRs). These PRR are expressed on both the surface [e.g. Toll-like receptor (TLR)-4] and in the cytoplasm [e.g. nucleotide-binding oligomerization domain (Nod)-like receptors] of cells of the innate immune system, primarily macrophages and dendritic cells. These cells initiate and propagate an inflammatory response by stimulating the synthesis and release of a variety of cytokines. Once an infection has occurred in the periphery, both cytokines and bacterial toxins deliver this information to the brain using both humoral and neuronal routes of communication. For example, binding of PRR can lead to activation of the afferent vagus nerve, which communicates neuronal signals via the lower brain stem (nucleus tractus solitarius) to higher brain centers such as the hypothalamus and amygdala. Blood-borne cytokines initiate a cytokine response from vascular endothelial cells that form the blood–brain barrier (BBB). Cytokines can also reach the brain directly by leakage through the BBB via circumventricular organs or by being synthesized within the brain, thus forming a mirror image of the cytokine milieu in the periphery. Although all cells within the brain are capable of initiating cytokine secretion, microglia have an early response to incoming neuronal and humoral stimuli. Inhibition of proinflammatory cytokines that are induced following bacterial infection blocks the appearance of sickness behaviors. Collectively, these data are consistent with the notion that the immune system communicates with the brain to regulate behavior in a way that is consistent with animal survival.

IL-1β impairs insulin-like growth factor I-induced differentiation and downstream activation signals of the insulin-like growth factor I receptor in myoblasts

Proinflammatory cytokines are elevated in disorders characterized by muscle wasting and weakness, such as inflammatory myopathies and AIDS wasting. We recently demonstrated that TNF-α impairs the ability of insulin-like growth factor (IGF)-I to promote protein synthesis in muscle precursor cells. In this study we extend these findings by showing that low concentrations of IL-1β impair IGF-I-dependent differentiation of myoblasts, as assessed by expression of the muscle specific protein, myosin heavy chain. In the absence of exogenous IGF-I, IL-1β (1 ng/ml) did not impair muscle cell development. However, in the presence of IGF-I, 100-fold lower concentrations of IL-1β (0.01 ng/ml) significantly suppressed myoblast differentiation, protein synthesis, and myogenin expression. Increasing IL-1β to 1 ng/ml completely blocked the anabolic actions of IGF-I in murine C2C12 myoblasts. Similarly, IL-1β inhibited IGF-I-stimulated protein synthesis in primary porcine myoblasts. IL-1β impaired the actions of IGF-I at a point distal to the IGF receptor, and this was not due to IL-1β-induced cell death. Instead, IL-1β inhibited the ability of IGF-I to phosphorylate tyrosine residues on both of its downstream docking proteins, insulin receptor substrate 1 and insulin receptor substrate 2. These data establish that physiological concentrations of IL-1β block the ability of IGF-I to promote protein synthesis, leading to reduced expression of the myogenic transcription factor, myogenin, and the subsequent development of more mature differentiated cells that express myosin heavy chain. Collectively, the results are consistent with the notion that very low concentrations of IL-1β significantly impair myogenesis, but they are unable to do so in the absence of the growth factor IGF-I.

Central administration of insulin-like growth factor-I decreases depressive-like behavior and brain cytokine expression in mice

Exogenous administration of insulin-like growth factor (IGF)-I has anti-depressant properties in rodent models of depression. However, nothing is known about the anti-depressant properties of IGF-I during inflammation, nor have mechanisms by which IGF-I alters behavior following activation of the innate immune system been clarified. We hypothesized that central IGF-I would diminish depressive-like behavior on a background of an inflammatory response and that it would do so by inducing expression of the brain-derived neurotrophic factor (BDNF) while decreasing pro-inflammatory cytokine expression in the brain. IGF-I (1,000 ng) was administered intracerebroventricularly (i.c.v.) to CD-1 mice. Mice were subsequently given lipopolysaccharide i.c.v. (LPS, 10 ng). Sickness and depressive-like behaviors were assessed followed by analysis of brain steady state mRNA expression. Central LPS elicited typical transient signs of sickness of mice, including body weight loss, reduced feed intake and decreased social exploration toward a novel juvenile. Similarly, LPS increased time of immobility in the tail suspension test (TST). Pretreatment with IGF-I or antidepressants significantly decreased duration of immobility in the TST in both the absence and presence of LPS. To elucidate the mechanisms underlying the anti-depressant action of IGF-I, we quantified steady-state mRNA expression of inflammatory mediators in whole brain using real-time RT-PCR. LPS increased, whereas IGF-I decreased, expression of inflammatory markers interleukin-1ß (IL-1ß), tumor necrosis factor-(TNF)α, inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP). Moreover, IGF-I increased expression of BDNF. These results indicate that IGF-I down regulates glial activation and induces expression of an endogenous growth factor that shares anti-depressant activity. These actions of IGF-I parallel its ability to diminish depressive-like behavior.

Regulation of myeloid growth and differentiation by the insulin-like growth factor I receptor

Flow cytometry was used to examine the expression of type I insulin-like growth factor receptors (IGF-IR) on three types of human hematopoietic cells that represent different stages of myeloid lineage development. Both HL-60 (promyeloid) and U-937 (monocytic) cells express abundant IGF-IR protein (>79% cells positive for the IGF-IR), whereas KG-1 myeloblasts express negligible levels of IGF-IR (<1% IGF-IR-positive cells). Exogenous IGF-I, IGF-II, and an IGF-I analog that binds poorly to IGF-binding protein-3 (des-IGF-I) increased DNA synthesis of HL-60 and U-937 cells in a dose-dependent (1–25 ng/ml) fashion by 2- to 4-fold in serum-free medium, whereas KG-1 cells did not respond to any of these growth factors. The IGF-induced increase in proliferation of HL-60 promyeloid cells was inhibited by soluble IGF-binding protein-3 (500 ng/ml) when these cells were stimulated with 10 ng/ml of either IGF-I (53 ± 8%) or IGF-II (59 ± 8%), but not with des-IGF-I (3 ± 1%). In contrast, the anti-IGF-IR monoclonal antib...

Voluntary wheel running, but not a diet containing (-)-epigallocatechin-3-gallate and β-alanine, improves learning, memory and hippocampal neurogenesis in aged mice

Aging is associated with impaired learning and memory accompanied by reductions in adult hippocampal neurogenesis and brain expression of neurotrophic factors among other processes. Epigallocatechin-3-gallate (EGCG, a green tea catechin), β-alanine (β-ala, the precursor of carnosine), and exercise have independently been shown to be neuroprotective and to reduce inflammation and oxidative stress in the central nervous system. We hypothesized that EGCG, β-ala supplementation or exercise alone would improve learning and memory and increase neurogenesis in aged mice, and the combined intervention would be better than either treatment alone. Male Balb/cByJ mice (19 months) were given AIN-93M diet with or without EGCG (182mg/kg/d) and β-ala (417mg/kg/d). Half of the mice were given access to a running wheel (VWR). The first 10 days, animals received 50mg/kg bromodeoxyuridine (BrdU) daily. After 28 days, learning and memory was assessed by Morris water maze (MWM) and contextual fear conditioning (CFC). Brains were collected for immunohistochemical detection of BrdU and quantitative mRNA expression in the hippocampus. VWR increased the number of BrdU cells in the dentate gyrus, increased expression of brain-derived neurotrophic factor, decreased expression of the inflammatory cytokine interleukin-1β, and improved performance in the MWM and CFC tests. The dietary intervention reduced brain oxidative stress as measured by 4-hydroxynonenal in the cerebellum, but had no effect on BrdU labeling or behavioral performance. These results suggest that exercise, but not a diet containing EGCG and β-ala, exhibit pro-cognitive effects in aged mice when given at these doses in this relatively short time frame.

Interleukin-1 beta converting enzyme is necessary for development of depression-like behavior following intracerebroventricular administration of lipopolysaccharide to mice

BackgroundInterleukin-1 beta converting enzyme (ICE, caspase 1) is a cysteine protease that processes immature pro-IL-1β into active mature IL-1β. IL-1β is a pro-inflammatory cytokine that mediates many of the physiological and behavioral responses to inflammation. Genetic deletion of ICE has previously been shown to prevent some negative physiologic responses to lipopolysaccharide (LPS)-induced inflammation.MethodsHere we used a preclinical murine model to test the hypothesis that ICE is necessary for development of depression-like behaviors following intracerebroventricular (ICV) treatment with LPS. Adult male ICE knockout (ICE KO) and congenic wild-type C57BL/6 J (WT) mice were administered LPS either ICV at 100 ng/mouse or intraperitoneally (IP) at 830 μg/kg body weight or an equal volume of saline as controls. Mice were monitored up to 48 h after treatment for both sickness and depression-like behaviors.ResultsLPS given ICV induced a loss of body weight in both WT and ICE KO mice. This sickness response was similar between WT and ICE KO mice. As expected, LPS administered ICV increased immobility in the forced swim test (FST) and decreased sucrose preference in WT mice but no change in either of these two depression-like behaviors was observed in ICE KO mice. Expression of TNF-α and CD11b in brain was lower in ICE-KO mice at 24 h following ICV administration of LPS compared to WT mice. In contrast, when LPS was given systemically, sickness response, depression-like behaviors, and expression of these genes were similar between the two strains of mice.ConclusionsThese findings indicate that ICE plays a specific role in depression-like behavior induced by a central inflammatory stimuli even though it is not required when LPS is administered systemically.

Insulin-like growth factor-I enhances the biological activity of brain-derived neurotrophic factor on cerebrocortical neurons

Insulin-like growth factor (IGF)-I and brain-derived neurotrophic factor (BDNF) act within the brain to enhance neuronal survival and plasticity. We extend these findings by showing that the presence of both neurotrophins is required to depress the rise in intracellular Ca2+ caused by glutamate in primary cultures of cerebrocortical neurons. IGF-I enhanced expression of BDNF receptors (Trk-B) and increased the ability of BDNF to induce ERK1/2 phosphorylation. This IGF-I-induced increase in BDNF responsiveness describes a new interaction between these peptides in the brain.

Insulin-like growth factor-I peptides act centrally to decrease depression-like behavior of mice treated intraperitoneally with lipopolysaccharide

Centrally administered insulin-like growth factor (IGF)-I has anti-depressant activity in several rodent models, including lipopolysaccharide (LPS)-induced depression. In this study we tested the ability of IGF-I and GPE (the N-terminal tri-peptide derived from IGF-I) to alter depression-like behavior induced by intraperitoneal (i.p.) administration of LPS in a preventive and curative manner. In the first case, IGF-I (1 μg) or GPE (5 μg) was administered i.c.v. to CD-1 mice followed 30 min later by 330 μg/kg body weight i.p. LPS. In the second case, 830 μg/kg body weight LPS was given 24 h prior to either IGF-I or GPE. When administered i.p., LPS induced full-blown sickness assessed as a loss of body weight, decrease in food intake and sickness behavior. None of these indices were affected by IGF-I or GPE. LPS also induced depression-like behavior; assessed as an increased duration of immobility in the tail suspension and forced swim tests. When administered before or after LPS, IGF-I and GPE abrogated the LPS response; attenuating induction of depression-like behaviors and blocking preexistent depression-like behaviors. Similar to previous work with IGF-I, GPE decreased brain expression of cytokines in response to LPS although unlike IGF-I, GPE did not induce the expression of brain-derived neurotrophic factor (BDNF). LPS induced expression of tryptophan dioxygenases, IDO1, IDO2 and TDO2, but expression of these enzymes was not altered by GPE. Thus, both IGF-I and GPE elicit specific improvement in depression-like behavior independent of sickness, an action that could be due to their anti-inflammatory properties.

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation.

The components of the insulin‐like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco‐2, have been investigated. Caco‐2 cells proliferated in serum‐free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF‐I (10 nM) increased Caco‐2 cell growth in serum‐free medium, but not to the rate seen with serum. Multiple IGF‐II mRNA species were produced by Caco‐2 cells, but IGF‐I mRNA was undetectable. Secretion of radioimmunoassayable IGF‐II corresponded with steady‐state levels of IGF‐II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco‐2 cell differentiation. Levels of sucrase‐isomaltase mRNA, a marker for enterocytic differentiation, increased 12‐fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum‐free conditioned medium revealed that Caco‐2 cells produce several IGF binding proteins (IGFBPs), including IGFBP‐2, ‐3, and ‐4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco‐2 cell differentiation: IGFBP‐3 and IGFBP‐2 increased 8.5‐fold and 5‐fold, respectively, whereas IGFBP‐4 and the 31,000 M, species decreased 43% and 90%. Caco‐2 cell clones stably transfected with a human IGFBP‐4 cDNA construct exhibited a 60% increase in steady‐state level of IGFBP‐4 mRNA, and secreted twice as much IGFBP‐4 protein as controls. Moreover, IGFBP‐4‐overexpressing cells proliferated at only 25% the rate of control cells in serum‐free medium, in conjunction with a 70% increase in expression of sucrase‐isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco‐2 cell proliferation and differentiation.

Tumor Necrosis Factor α Inhibits Cyclin A Expression and Retinoblastoma Hyperphosphorylation Triggered by Insulin-like Growth Factor-I Induction of New E2F-1 Synthesis

Cyclin A is required for cell cycle S phase entry, and its overexpression contributes to tumorigenesis. Release of pre-existing E2Fs from inactive complexes of E2F and hypophosphorylated retinoblastoma (RB) is the prevailing dogma for E2F transcriptional activation of target genes such as cyclin A. Here we explored the hypothesis that new synthesis of E2F-1 is required for insulin-like growth factor-I (IGF-I) to induce cyclin A accumulation and RB hyperphosphorylation, events that are targeted by tumor necrosis factor α (TNFα) to arrest cell cycle progression. We first established that IGF-I increases expression of cyclin A, causes hyperphosphorylation of RB, and augments the mass of E2F-1 in a time-dependent manner. As expected, E2F-1 small interfering RNA blocks the ability of IGF-I to increase synthesis of E2F-1. Most important, this E2F-1 small interfering RNA also blocks the ability of IGF-I to increase cyclin A accumulation and to hyperphosphorylate RB. We next established that TNFα dose-dependently inhibits IGF-I-induced phosphorylation of both RB and histone H1 by cyclin A-dependent cyclin-dependent kinases. Cyclin-dependent kinase 2 (Cdk2) mediates this suppression because co-immunoprecipitation experiments revealed that TNFα reduces the amount of IGF-I-induced cyclin A that binds Cdk2, leading to a reduction in Cdk2 enzymatic activity. TNFα antagonizes the ability of IGF-I to increase mass of both E2F-1 and cyclin A but not cyclin E or D1. The cytostatic property of TNFα is also shown by its ability to block IGF-I-stimulated luciferase activity of a cyclin A promoter reporter. Deletion of an E2F recognition site from this reporter eliminates the regulatory effects of both IGF-I and TNFα on cyclin A transcription, indicating the essential role of E2F-1 in mediating their cross-talk. Collectively, these results establish that TNFα targets IGF-I-induced E2F-1 synthesis, leading to inhibition of the subsequent accumulation in cyclin A, formation of cyclin A-Cdk2 complexes, hyperphosphorylation of RB, and cell cycle arrest.