Leptin modulates the late fever response to LPS in diet-induced obese animals.

Leptin is an important modulator of both inflammation and energy homeostasis, making it a key interface between the inflammatory response to pathogenic stimuli and the energy status of the host. In previous studies we demonstrated that sickness responses to systemic immune challenge, including fever, are significantly exacerbated in diet induced obese animals. To investigate whether this exacerbation is functionally linked to the obesity associated increase in circulating levels of leptin, a species-specific leptin antiserum (LAS) was used to neutralize endogenous leptin in diet-induced obese adult male Wistar rats treated with a single intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) (100µg/kg). LAS significantly reduced the magnitude of the later phases of the fever response, and attenuated the circulating levels of IL-6, IL-1ra and bioactivity of leptin in the obese animals. In addition, the antiserum significantly attenuated the hypothalamic expression of IL-1ß, IκBα, COX2, SOCS3 and IL-6 in both lean and obese rats 10h after the LPS injection and NF-IL6 in the hypothalamus of obese rats only. The relatively late rise in brain IL-6 suggested a role in mediating the extended fever response in obese animals and we tested this by neutralizing brain IL-6 using an IL6-AS injected intracerebroventricularly (4µl, icv). The IL6-AS significantly but transiently (between 9h and 12h post LPS) reduced the late fever response of obese rats. These results demonstrate that leptin plays an important part in modulating the late portion of the fever response to LPS, likely through the induction of hypothalamic IL-6 in obese animals.

Leptin is involved in age-dependent changes in response to systemic inflammation in the rat.

Obesity contributes to a state of subclinical peripheral and central inflammation and is often associated with aging. Here we investigated the source and contribution of adipose tissue derived cytokines and the cytokine-like hormone leptin to age-related changes in lipopolysaccharide (LPS)-induced brain-controlled sickness-responses. Old (24 months) and young (2 months) rats were challenged with LPS or saline alone or in combination with a neutralizing leptin antiserum (LAS) or control serum. Changes in the sickness-response were monitored by biotelemetry. Additionally, ex vivo fat-explants from young and old rats were stimulated with LPS or saline and culture medium collected and analyzed by cytokine-specific bioassays/ELISAs. We found enhanced duration/degree of the sickness-symptoms, including delayed but prolonged fever in old rats. This response was accompanied by increased plasma-levels of interleukin (IL)-6 and IL-1ra and exaggerated expression of inflammatory markers in brain and liver analyzed by RT-PCR including inhibitor κBα, microsomal prostaglandin synthase and cyclooxygenase 2 (brain). Moreover, for the first time, we were able to show prolonged elevated plasma leptin-levels in LPS-treated old animals. Treatment with LAS in young rats tended to attenuate the early- and in old rats the prolonged febrile response. Fat-explants exhibited unchanged IL-6 but reduced IL-1ra and tumor necrosis factor (TNF)-α release from adipose tissue of aged compared to young animals. In addition, we found increased expression of the endogenous immune regulator microRNA146a in aged animals suggesting a role for these mediators in counteracting brain inflammation. Overall, our results indicate a role of adipose tissue and leptin in "aging-related-inflammation" and age-dependent modifications of febrile-responses.

The effects of dietary saturated fat on basal hypothalamic neuroinflammation in rats.

Recent evidence has demonstrated that consumption of high fat diets can trigger brain inflammation and subsequent injury in the absence of any peripheral inflammatory signaling. Here we sought to investigate whether a link exists between the concentration of highly saturated fats in the diet and the development of inflammation in the brain of rats and, whether the source of the saturated fat was an important factor in this process. Adult male rats had access to diets with a moderate level of total fat (32% of calories as fat) varying in level of saturated fat [low (20%) vs high (>60%)] and its source (butter or coconut oil). After 8 weeks of diet exposure peripheral and central tissues were collected for analysis of inflammatory signals. Neither blood nor white adipose tissue exhibited any changes in inflammatory mediators regardless of the saturated fat content or the source. In the brain however, we observed significant hypothalamic upregulation of the expression of markers of glial activation as well as of interleukin (IL)-1,6 and nuclear factor (NF)-IL-6, which were highest in the group fed the butter-based diets. The increase in these inflammatory mediators had no effect on basal body temperature or the temperature response to systemic lipopolysaccharide (LPS). The present results indicate that hypothalamic inflammation associated with consumption of diets high in fat is directly linked to the saturated fat content as well as the source of that fat. These effects are likely linked to other pathophysiological changes in the regulation of metabolism.

Interleukin-10 modulates the synthesis of inflammatory mediators in the sensory circumventricular organs: implications for the regulation of fever and sickness behaviors.

BACKGROUND: Whereas the role played by interleukin (IL)-10 in modulating fever and sickness behavior has been linked to it targeting the production of pro-inflammatory cytokines in the circulation, liver and spleen, it is not known whether it could directly target the local production of pro-inflammatory cytokines within the sensory circumventricular organs (CVOs) situated within the brain, but outside the blood-brain barrier. Using inactivation of IL-10, we, therefore, investigated whether IL-10 could modulate the synthesis of pro-inflammatory cytokines within the sensory CVOs, in particular the organum vasculosum laminae terminalis (OVLT) and area postrema (AP). FINDINGS: Primary OVLT and AP microcultures were established from topographically excised rat pup brain tissue. The microcultures were pretreated with either IL-10 antibodies (AB) (10 µl/350 µl medium) or phosphate-buffered saline (PBS) (10 µl/350 µl medium) before being incubated with lipopolysaccharide (LPS) (100 µg/ml) or PBS in complete medium for 6 h. Supernatants were removed from the microcultures after 6 h of incubation with LPS and used for the determination of IL-6 and tumor necrosis factor (TNF)-α. Pre-treating the OVLT and AP microcultures with IL-10 antibodies significantly enhanced the LPS-induced increase in TNF-α and IL-6 in the supernatant obtained from the microcultures. CONCLUSIONS: Our results show for the first time that the LPS-induced release of pro-inflammatory cytokines in cells cultured from the AP and OVLT can be modulated in the presence of IL-10 antibodies. Thus, we have identified that the sensory CVOs may have a key role to play in both the initiation and modulation of neuroinflammation.

Effects of endogenous glucocorticoid secretion on the interleukin-6 response to bacterial endotoxin in pregnant and non-pregnant rats.

Glucocorticoids (GCs) are released in response to immune activation by the bacterial endotoxin, lipopolysaccharide (LPS). However, GC secretion in response to immune activation and other stressors is attenuated at term of pregnancy. GCs are important modulators of the immune response, and both pro- and anti-inflammatory effects are described. Here, we examined whether GC secretion in response to LPS is maintained in earlier pregnancy before term, and investigated the role of endogenous GCs in modulating LPS-induced circulating cytokines, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), in pregnant compared to non-pregnant female rats. Plasma corticosterone (Cort) and ACTH responses to LPS were well maintained in pregnant rats at embryonic days 15/16 (E15/16) and E18/19 compared to non-pregnant rats. At E19, maternal LPS administration increased fetal plasma Cort and decreased testosterone in male fetuses. In non-pregnant animals, pretreatment with the GC synthesis inhibitor, metyrapone, inhibited the LPS-induced increase in IL-6, and the IL-6 response was restored by Cort replacement, indicating that LPS induction of IL-6 is Cort-dependent. In E15 pregnant animals, metyrapone had no effect on LPS-induced IL-6 levels, indicating that LPS-induction of IL-6 is not dependent on Cort. These contrasting patterns of IL-6 induction in non-pregnant and pregnant animals were reflected in levels of hypothalamic Socs3 mRNA, an indicator of IL-6 signaling pathway activation. In both non-pregnant and pregnant rats, LPS-induced plasma TNF-α responses were inhibited by metyrapone but not re-instated by Cort replacement. It is suggested that altered GC regulation of IL-6 may be required to sustain specialized functions of IL-6 during pregnancy.

Spatiotemporal nuclear factor interleukin-6 expression in the rat brain during lipopolysaccharide-induced fever is linked to sustained hypothalamic inflammatory target gene induction.

Rats injected with lipopolysaccharide (LPS) show brain-controlled sickness symptoms, including fever. In these animals, early genomic activation of brain cells was previously monitored by immunohistochemical detection of transcription factors such as nuclear factor (NF)-κB or signal transducer and activator of transcription (STAT)3 and was linked to the initiation or maintenance of the febrile response. To investigate whether NF-IL6 might be another important transcription factor implicated in this kind of immune-to-brain signaling, rats were injected with LPS (100 µg/kg, intraperitoneally) or phosphate-buffered saline, and brains were analyzed by immunohistochemistry, real-time PCR, or Western blot 4, 6, 8, and 10 hours later. Moderate to strong LPS-induced nuclear NF-IL6 immunoreactivity (IR) occurred in a time-dependent manner within circumventricular organs, namely, the vascular organ of the lamina terminalis, the subfornical organ, the area postrema, and the median eminence, brain structures with a leaky blood-brain barrier. Furthermore, nuclear NF-IL6-IR was observed in the pituitary gland, the choroid plexus, and the meninges as well as blood vessels throughout the entire brain. Endothelial, microglial, and ependymal cells, astrocytes, perivascular macrophages, and neurons exhibited LPS-induced nuclear NF-IL6-IR; mRNA levels of NF-IL6, responsive inflammatory genes, and NF-IL6 protein levels were significantly elevated. As opposed to observations on STAT3 or NFκB, the percentage of NF-IL6-reactive cells increased in parallel to late phases of the febrile response. In conclusion, these results suggest a potential role for NF-IL6 in the maintenance or possibly the termination of LPS-induced fever. Moreover, we propose NF-IL6 to be a delayed brain cell activation marker.

Acute starvation alters lipopolysaccharide-induced fever in leptin-dependent and -independent mechanisms in rats.

A decrease in leptin levels with the onset of starvation triggers a myriad of physiological responses including immunosuppression and hypometabolism/hypothermia, both of which can counteract the fever response to pathogens. Here we examined the role of leptin in LPS-induced fever in rats that were fasted for 48 h prior to inflammation with or without leptin replacement (12 µg/day). The preinflammation fasting alone caused a progressive hypothermia that was almost completely reversed by leptin replacement. The LPS (100 µg/kg)-induced elevation in core body temperature (T(core)) was attenuated in the fasted animals at 2-6 h after the injection, an effect that was not reversed by leptin replacement. Increasing the LPS dose to 1,000 µg/kg caused a long-lasting fever that remained unabated for up to 36 h after the injection in the fed rats. This sustained response was strongly attenuated in the fasted rats whose T(core) started to decrease by 18 h after the injection. Leptin replacement almost completely restored the prolonged fever. The attenuation of the prolonged fever in the fasted animals was accompanied by the diminution of proinflammatory PGE(2) in the cerebrospinal fluid and mRNA of proopiomelanocortin (POMC) in the hypothalamus. Leptin replacement prevented the fasting-induced reduction of POMC but not PGE(2). Moreover, the leptin-dependent fever maintenance correlated closely with hypothalamic POMC levels (r = 0.77, P < 0.001). These results suggest that reduced leptin levels during starvation attenuate the sustained fever response by lowering hypothalamic POMC tone but not PGE(2) synthesis.

Prenatal inflammation-induced hypoferremia alters dopamine function in the adult offspring in rat: relevance for schizophrenia.

Maternal infection during pregnancy has been associated with increased incidence of schizophrenia in the adult offspring. Mechanistically, this has been partially attributed to neurodevelopmental disruption of the dopamine neurons, as a consequence of exacerbated maternal immunity. In the present study we sought to target hypoferremia, a cytokine-induced reduction of serum non-heme iron, which is common to all types of infections. Adequate iron supply to the fetus is fundamental for the development of the mesencephalic dopamine neurons and disruption of this following maternal infection can affect the offspring's dopamine function. Using a rat model of localized injury induced by turpentine, which triggers the innate immune response and inflammation, we investigated the effects of maternal iron supplementation on the offspring's dopamine function by assessing behavioral responses to acute and repeated administration of the dopamine indirect agonist, amphetamine. In addition we measured protein levels of tyrosine hydroxylase, and tissue levels of dopamine and its metabolites, in ventral tegmental area, susbtantia nigra, nucleus accumbens, dorsal striatum and medial prefrontal cortex. Offspring of turpentine-treated mothers exhibited greater responses to a single amphetamine injection and enhanced behavioral sensitization following repeated exposure to this drug, when compared to control offspring. These behavioral changes were accompanied by increased baseline levels of tyrosine hydroxylase, dopamine and its metabolites, selectively in the nucleus accumbens. Both, the behavioral and neurochemical changes were prevented by maternal iron supplementation. Localized prenatal inflammation induced a deregulation in iron homeostasis, which resulted in fundamental alterations in dopamine function and behavioral alterations in the adult offspring. These changes are characteristic of schizophrenia symptoms in humans.

Changes in hypothalamically mediated acute-phase inflammatory responses to lipopolysaccharide in diet-induced obese rats.

Recent evidence suggests that inflammation may be a common underlying cause of many obesity-associated conditions. To test whether obesity changes the response to inflammation, we investigated its effects on the acute phase of the inflammatory response to an endogenous pathogen, lipopolysaccharide (LPS). Diet-induced obese male Wistar rats exhibited an increased and prolonged fever response to LPS (100 microg/kg) relative to lean rats. LPS-treated obese rats also showed a greater increase in circulating TNF-alpha, IL-6, and IL-1 receptor antagonist within the first 8 h after LPS injection. LPS induced an increase in circulating leptin only in obese rats with no effect in lean rats. Analysis of expression of pyrogenic signaling in the hypothalamus demonstrated that obese rats show a greater increase in IL-1beta peaking at 2 h after LPS injection and suppressor of cytokine signaling 3 and IL-6 peaking at the 8-h time point. LPS-treated obese rats showed a significantly higher expression of IL-1 receptor antagonist in white adipose tissue (WAT) than lean rats, and WAT from obese rats incubated in LPS-supplemented medium (100 ng/ml) secreted a significantly higher level of IL-6. Overall, these results suggest that diet-induced obesity induces changes in the inflammatory response rendering the obese rats more responsive to the effects of LPS. These data also support the hypothesis that qualitative changes in WAT associated with obesity may contribute to these effects.

Attenuated fever in rats during late pregnancy is linked to suppressed interleukin-6 production after localized inflammation with turpentine.

An attenuated fever response to pathogens during late pregnancy is a phenomenon that has been described in several mammalian species, and although mechanisms are not completely understood, decreased prostaglandin E2 (PGE2) synthesis has been implicated. Upstream of PGE2, there is evidence to suggest that anti-inflammatory cytokines such as interleukin-1 receptor antagonist (IL-1ra) could play a significant role. In the present study we addressed the role of pro-inflammatory cytokines during late pregnancy, specifically interleukin-6 (IL-6), an important circulating mediator in fever. Turpentine oil (TURP), a very potent pyrogen and activator of IL-6, was injected into the hind-limb muscle of rats at the 18th day of pregnancy (GD 18) or in non-pregnant (NP) age-matched female controls. As expected, TURP injection induced a highly significant fever in the NP animals, which peaked 11 h post-injection and lasted for over 24 h. This was accompanied by a significant rise in circulating IL-6 levels, which correlated with changes in PGE2 synthesizing enzymes expression in the hypothalamus. In complete contrast, TURP-induced fever was totally absent in GD 18 animals whose body temperature did not deviate from basal values. The lack of response was additionally reflected by the absence of change in IL-6 concentration and by the significant attenuation of PGE2 synthesizing enzymes expression, which correlated with the suppressed expression of SOCS3, a hypothalamic marker of IL-6 activity. Contrary to the changes in circulating IL-6 levels at GD 18, IL-1ra was induced to levels comparable to those of NP females, suggesting that the influence of this anti-inflammatory cytokine on the fever response to TURP is at best minimal. These data further confirm the importance of IL-6 in fever generation and provide evidence that it may be a key component of the attenuated fever response in late pregnancy.

Effects of prenatal infection on prepulse inhibition in the rat depend on the nature of the infectious agent and the stage of pregnancy.

Maternal infection during pregnancy is a risk factor for some psychiatric illnesses of neurodevelopmental origin such as schizophrenia and autism. In experimental animals, behavioral and neuropathological outcomes relevant to schizophrenia have been observed in offspring of infected dams. However, the type of infectious agent used and gestational age at time of administration have varied. The objective of the present study was to compare the effects of prenatal challenge with different immune agents given at different time windows during gestation on behavioral outcomes in offspring. For this, pregnant rats were administered bacterial endotoxin (lipopolysaccharide, LPS), the viral mimic polyinosinic: polycytidylic acid (poly I:C), or turpentine, an inducer of local inflammation, at doses known to produce fever, at three different stages in pregnancy: embryonic day (E)10-11, E15-16 and E18-19. Prepulse inhibition of acoustic startle (PPI) was later measured in male adult offspring. PPI was significantly decreased in offspring after prenatal LPS treatment at E15-16 and E18-19. Intramuscular injection of pregnant dams with turpentine at E15-16 also decreased PPI in adult offspring. Maternal poly I:C administration had no significant effect on PPI in offspring. In contrast to prenatal LPS exposure, acute LPS administration to naive adult males had no effect on PPI. Thus, prenatal exposure both to a systemic immunogen and to local inflammation at brief periods during later pregnancy produced lasting deficits in PPI in rat offspring. These findings support the idea that maternal infection during critical windows of pregnancy could contribute to sensorimotor gating deficits in schizophrenia.

Leptin induces cyclooxygenase-2 via an interaction with interleukin-1beta in the rat brain.

In addition to its central effects on appetite regulation, leptin has been implicated in immune function and inflammation. Previous data suggested that leptin acts as an inflammatory signal within the brain, as exogenously administered leptin induced fever, a typical brain-regulated inflammatory response. The present study aimed to delineate the inflammatory actions and cellular targets of leptin in the brain by examining its effects on the expression of interleukin (IL)-1beta and cyclooxygenase (COX)-2, two important inflammatory components of the fever response. Intracerebroventricular injection of leptin (5 microg/rat) induced IL-1beta and COX-2 mRNA and protein in the hypothalamus between 1 and 3 h after treatment as determined by reverse transcription-polymerase chain reaction and immunohistochemistry. Coinjection of IL-1 receptor antagonist (100 microg/rat, intracerebroventricular) attenuated leptin-induced COX-2, whereas IL-1 receptor antagonist had no effect on endogenous IL-1beta levels, suggesting that leptin induces COX-2 via, at least partly, IL-1beta action. IL-1beta protein expression was induced in macrophages in the meningis and perivascular space after leptin treatment, whereas COX-2 induction was observed in endothelial cells, indicating the roles for these non-neuronal cells in mediating inflammatory actions of leptin. In addition, neutralization of endogenous circulating leptin with anti-leptin antiserum attenuated intraperitoneal lipopolysaccharide (100 microg/kg)-induced brain IL-1beta and COX-2 upregulation, suggesting that leptin indeed acts as an inflammatory signal to the brain during systemic inflammation. These findings are in contrast to the effects of leptin on appetite regulation where it is believed to act primarily on neurons, thus presenting a distinct anatomical basis for the inflammatory and appetite regulatory actions of leptin in the brain.

Circulating leptin mediates lipopolysaccharide-induced anorexia and fever in rats.

Anorexia and fever are important features of the host's response to inflammation that can be triggered by the bacterial endotoxin lipopolysaccharide (LPS) and the appetite suppressant leptin. Previous studies have demonstrated that LPS induces leptin synthesis and secretion in the periphery, and that the action of leptin on appetite suppression and fever are dependent on brain interleukin (IL)-1beta. However, the role of leptin as a neuroimmune mediator of LPS-induced inflammation has not been fully elucidated. To address this issue, we neutralized circulating leptin using a leptin antiserum (LAS) and determined how this neutralization affected LPS-induced anorexia, fever and hypothalamic IL-1beta. Adult male rats were separated into four treatment groups, namely LPS + normal sheep serum (NSS), LPS + LAS, saline + LAS and saline + NSS. Intraperitoneal injection of LPS (100 microg kg(-1)) induced a significant reduction in food intake and body weight, which were significantly reversed in the presence of LAS (1 ml kg(-1)), 8 and 24 h after treatment. In addition, LPS-induced fever was significantly attenuated by LAS over the duration of the fever response (8 h). Lipopolysaccharide induced an increase of circulating IL-6, another potential circulating pyrogen, which was not affected by neutralization of leptin at 2 h. Interleukin-1beta mRNA at 1 and 8 h, and IL-1 receptor antagonist (ra) at 2 h were significantly upregulated in the hypothalamus of LPS-treated animals. The induction of these cytokines was attenuated in the presence of LAS. These results are the first to demonstrate that leptin is a circulating mediator of LPS-induced anorexia and fever, probably through a hypothalamic IL-1beta-dependent mechanism.