Repeated Lipopolysaccharide Administration Research Articles

Effects of hindgut acidosis on production, metabolism, and inflammatory biomarkers in previously immune-activated lactating dairy cows

Previous stressors and systemic inflammation may increase the intestine's susceptibility to hindgut acidosis (HGA). Therefore, our experimental objectives were to evaluate the effects of isolated HGA on metabolism, production, and inflammation in simultaneously immune-activated lactating cows. Twelve rumen-cannulated Holstein cows (118 ± 41 d in milk; 1.7 ± 0.8 parity) were enrolled in a study with 3 experimental periods (P). Baseline data were collected during P1 (5 d). On d 1 of P2 (2 d), all cows received an i.v. lipopolysaccharide (LPS) bolus (0.2 µg/kg of body weight; BW). During P3 (4 d), cows were randomly assigned to 1 of 2 abomasal infusion treatments: (1) control (LPS-CON; 6 L of H2O/d; n = 6) or (2) starch infused (LPS-ST; 4 kg of corn starch + 6 L of H2O/d; n = 6). Treatments were allocated into 4 equal doses (1.5 L of H2O or 1 kg of starch and 1.5 L of H2O, respectively) and administered at 0000, 0600, 1200, and 1800 h daily. Additionally, both treatments received i.v. LPS on d 1 and 3 of P3 (0.8 and 1.6 µg/kg of BW, respectively) to maintain an inflamed state. Effects of treatment, time, and their interaction were assessed. Repeated LPS administration initiated and maintained an immune-activated state, as indicated by increased circulating white blood cells (WBC), serum amyloid A (SAA), and LPS-binding protein (LBP) during P2 and P3 (29%, 3-fold, and 50% relative to P1, respectively) for both abomasal infusion treatments. Regardless of abomasal treatment, milk yield and dry matter intake were decreased throughout P2 and P3 but with lesser severity following each LPS challenge (54, 44, and 37%, and 49, 42, and 40% relative to baseline on d 1 of P2, d 1 and d 3 of P3, respectively). As expected, starch infusions markedly decreased fecal pH (5.56 at nadir vs. 6.57 during P1) and increased P3 fecal starch relative to LPS-CON (23.7 vs. 2.4% of dry matter). Neither LPS nor starch infusions altered circulating glucose, insulin, nonesterified fatty acids, or β-hydroxybutyrate, although LPS-ST cows had decreased blood urea nitrogen throughout P3 (16% relative to LPS-CON). Despite the striking reduction in fecal pH, HGA had no additional effect on circulating WBC, SAA, or LBP. Thus, in previously immune-activated dairy cows, HGA did not augment the inflammatory state, as indicated by a lack of perturbations in production, metabolism, and inflammatory biomarkers.

Pathological consequences of chronic olfactory inflammation on neurite morphology of olfactory bulb projection neurons

Chronic olfactory inflammation (COI) in conditions such as chronic rhinosinusitis significantly impairs the functional and anatomical components of the olfactory system. COI induced by intranasal administration of lipopolysaccharide (LPS) results in atrophy, gliosis, and pro-inflammatory cytokine production in the olfactory bulb (OB). Although chronic rhinosinusitis patients have smaller OBs, the consequences of olfactory inflammation on OB neurons are largely unknown. In this study, we investigated the neurological consequences of COI on OB projection neurons, mitral cells (MCs) and tufted cells (TCs). To induce COI, we performed unilateral intranasal administration of LPS to mice for 4 and 10 weeks. Effects of COI on the OB were examined using RNA-sequencing approaches and immunohistochemical analyses. We found that repeated LPS administration upregulated immune-related biological pathways in the OB after 4 weeks. We also determined that the length of TC lateral dendrites in the OB significantly decreased after 10 weeks of COI. The axon initial segment of TCs decreased in number and in length after 10 weeks of COI. The lateral dendrites and axon initial segments of MCs, however, were largely unaffected. In addition, dendritic arborization and AIS reconstruction both took place following a 10-week recovery period. Our findings suggest that olfactory inflammation specifically affects TCs and their integrated circuitry, whereas MCs are potentially protected from this condition. This data demonstrates unique characteristics of the OBs ability to undergo neuroplastic changes in response to stress.

Splenic anti-inflammatory reflex in immune tolerance.

The injection of repeated doses of lipopolysaccharide (LPS) results in attenuation of the immune response, which is an important mechanism to prevent deleterious long-term excessive inflammation. Brain-mediated mechanisms are involved in this endogenous anti-inflammatory effect, but nothing is known about the putative role of the splenic anti-inflammatory reflex (which has recently been described as a powerful mechanism involved in the suppression of immune response) during immune tolerance. Therefore, we tested the hypothesis that endotoxin tolerance is at least in part mediated by the splenic anti-inflammatory reflex. Body core temperature (Tb) was measured in rats previously submitted to splenectomy. Immune tolerance was induced by means of five consecutive LPS (100 μg/kg) intraperitoneal injections at 24-h intervals. In sham operated rats, we observed a significant reduction of the febrile response to repeated administration of LPS, which was not altered in rats submitted to splenectomy. Moreover, plasma pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6] and prostaglanding E2 (PGE2) surges besides preoptic PGE2 levels were observed after the first LPS administration but not in tolerant animals, and this pattern was kept the same in splenectomized rats. These data are consistent with the notion that the splenic anti-inflammatory reflex does not modulate immune tolerance in rats.

S76. Omega-3 Fatty Acid Deficiency Augments C-Reactive Protein Biosynthesis in Response to Repeated Lipopolysaccharide Administration in Rats

The pathoetiology of major depressive disorder (MDD) is associated with elevated biomarkers of systemic inflammation, including C-reactive protein (CRP), and lower red blood cell (RBC) omega-3 polyunsaturated fatty acid (n-3 PUFA) levels. The present study investigated the effects of dietary-induced alterations in n-3 PUFA biostatus on basal and stimulated CRP levels in rats.

Impact of repeated lipopolysaccharide administration on ovarian signaling during the follicular phase of the estrous cycle in post-pubertal pigs.

Increased circulating lipopolysaccharide (LPS) results from heat stress (HS) and bacterial infection, both of which are associated with reduced female fertility. Specific effects of low-level, repeated LPS exposure on the ovary are unclear, as many studies utilize a bolus model and/or high dosage paradigm. To better understand the effects of chronic LPS exposure on ovarian signaling and function, post-pubertal gilts (n = 20) were orally administered altrenogest for 14 d to synchronize the beginning of the follicular phase of the ovarian cycle. For 5 d after synchronization, gilts (163 ± 3 kg) received IV administration of LPS (0.1 µg/kg BW, n = 10) or saline (CT, n = 10) 4× daily. Blood samples were obtained on days 1, 3, and 5 of LPS treatment. Follicular fluid was aspirated from dominant follicles on day 5, and whole ovarian homogenate was used for transcript and protein abundance analysis via quantitative real-time PCR and western blotting, respectively. There were no treatment differences detected in rectal temperature on any day (P ≥ 0.5). Administering LPS increased plasma insulin (P < 0.01), LPS-binding protein (LBP; P < 0.01), and glucose (P = 0.08) on day 1, but no treatment differences thereafter were observed (P = 0.66). There were no treatment differences in follicular fluid concentration of LBP or 17β-estradiol (P = 0.42). Gilts treated with LPS had increased abundance of ovarian TLR4 protein (P = 0.01), but protein kinase B (AKT) and phosphorylated AKT (pAKT) were unchanged and no effect of LPS on components of the phosphatidylinositol 3 kinase (PI3K) pathway were observed. There was no impact of LPS on ovarian abundance of STAR or CYP19A1, nor ESR1, LDLR, CYP19A1, CYP17A1, or 3BHSD. In conclusion, repeated, low-level LPS administration alters inflammatory but not steroidogenic or PI3K signaling in follicular phase gilt ovaries.

Dl-3-n-Butylphthalide improves lipopolysaccharide-induced depressive-like behavior in rats: involvement of Nrf2 and NF-κB pathways.

Dl-3-n-Butylphthalide (NBP), a small molecule compound extracted from the seeds of Apium graveolens, possesses a large range of biological effects. Here, we attempted to explore the therapeutic effects of NBP on lipopolysaccharide (LPS)-induced major depressive disorder (MDD) and gain further insight into the underlying mechanisms of the antidepressant effects of NBP. We evaluated the effect of NBP against LPS-induced behavioral changes in rats. We also examined the inflammation, oxidative stress, and apoptosis markers and analyzed the Nrf2 and NF-κB pathways in the hippocampus of rats following repeated peripheral immune challenge by LPS for 2weeks (500μg/kg every other day). Our results indicated that repeated LPS administration induced the rats to a depressive-like state and activated inflammatory response, oxidative stress, and apoptosis reactions in the hippocampus. NBP treatment attenuated the LPS-induced abnormal behavior and ameliorated pathogenic processes in rats with MDD. NBP reduced the inflammatory response with inhibited expression of pro-inflammatory cytokines including IL-1β and IL-6 and downregulated the NF-κB signal pathway. Concurrent with the anti-inflammation action, NBP reduced LPS-induced oxidative reactions in the hippocampus and enhanced Nrf2-targeted signals, as evidenced by increased transcription of antioxidant enzymes and decreased malondialdehyde (MDA) production. In addition, NBP inhibited LPS-induced neuronal apoptosis in the rat brain, as evidenced by decreased apoptosis marker Caspase-3 production and TUNEL assay. These results provide more insight into pathogenesis of MDD and firstly demonstrated the potential antidepressant actions of NBP.

Fish oil supplementation attenuates neuroinflammation and alleviates depressive-like behavior in rats submitted to repeated lipopolysaccharide.

Depression is frequently associated with inflammation, whereas omega-3 polyunsaturated fatty acids (PUFAs) primarily found in fish oil possess anti-inflammatory properties. Although converging studies suggest an antidepressant effect of PUFAs, there is limited evidence directly linking the neuro-immune modulating features of PUFAs to the antidepressant actions. Therefore, we assessed the effects of fish oil (FO) supplementation on behavioral changes, inflammatory cytokine expression and oxidative reactions in frontal cortex and hippocampus of rats following repeated peripheral immune challenge by lipopolysaccharide (LPS) for 2weeks (500μg/kg every other day). Repeated LPS administration induced the rats to a depressive-like state and increased mRNA expression of pro-inflammatory cytokines, including 1L-1β, 1L-6 and TNF-α, in frontal cortex and hippocampus. FO supplementation attenuated the LPS-induced abnormal behavior and brain inflammatory response. Concurrent with the antidepressant action, FO also reduced LPS-induced oxidative reactions and neural apoptosis in the rat brain, as evidenced by decreased malondialdehyde (MDA) production, increased catalase activities and inhibited pro-apoptotic protein Bax mRNA expression. In addition, FO inhibited activation of NF-κB and iNOS induced by LPS. Interestingly, we found FO suppressed the activation of the inflammasome NLRP3 and ionotropic purinergic receptor P2X7R evoked by LPS, suggesting a potential anti-inflammatory mechanism for PUFAs. Besides, FO also restored the LPS-induced neurochemical disturbance, especially the balance between serotonin and kynurenine branches of tryptophan metabolism, which is tightly associated with depression. These findings provide novel insights into the antidepressant action of PUFAs and further strengthen the link between inflammation and depression.

Repeated LPS Injection Induces Distinct Changes in the Kynurenine Pathway in Mice.

The immune system has been recognized as a potential contributor to psychiatric disorders. In animals, lipopolysaccharide (LPS) is used to induce inflammation and behaviors analogous to some of the symptoms in these disorders. Recent data indicate that the kynurenine pathway contributes to LPS-induced aberrant behaviors. However, data are inconclusive regarding optimal LPS dose and treatment strategy. Here, we therefore aimed to evaluate the effects of single versus repeated administration of LPS on the kynurenine pathway. Adult C57BL6 mice were given 0.83mg/kg LPS as a single or a repeated injection (LPS+LPS) and sacrificed after 24, 48, 72, or 120h. Mice receiving LPS+LPS had significantly elevated brain kynurenine levels at 24 and 48h, and elevated serum kynurenine at 24, 48 and 72h. Brain kynurenic acid and quinolinic acid were significantly increased at 24 and 48h in mice receiving LPS+LPS, whereas serum kynurenic acid levels were significantly decreased at 24h. The increase of brain kynurenic acid by LPS+LPS was likely unrelated to the higher total dose as a separate group of mice receiving 1.66mg/kg LPS as single injection 24h prior to sacrifice did not show increased brain kynurenic acid. Serum quinolinic acid levels were not affected by LPS+LPS compared to vehicle. Animals given repeated injections of LPS showed a more robust induction of the kynurenine pathway in contrast to animals receiving a single injection. These results may be valuable in light of data showing the importance of the kynurenine pathway in psychiatric disorders.

Systematic Analysis of the Cytokine and Anhedonia Response to Peripheral Lipopolysaccharide Administration in Rats.

Inflammatory processes may cause depression in subsets of vulnerable individuals. Inflammation-associated behavioral changes are commonly modelled in rodents by administration of bacterial lipopolysaccharide (LPS). However, the time frame in which immune activation and depressive-like behavior occur is not very clear. In this study, we showed that systemic administration of LPS robustly increased circulating levels of corticosterone, leptin, pro- and anti-inflammatory cytokines, and chemokines. Serum concentrations of most analytes peaked within the first 6 h after LPS injection and returned to baseline values by 24 h. Chemokine levels, however, remained elevated for up to 96 h. Using an optimized sucrose preference test (SPT) we showed that sickness behavior was present from 2 to 24 h. LPS-induced anhedonia, as measured by decreased sucrose preference, lasted up to 96 h. To mimic the human situation, where depression develops after chronic inflammation, rats were preexposed to repeated LPS administration or subchronic restraint stress and subsequently challenged with LPS. While these procedures did not increase the duration of anhedonia, our results do indicate that inflammation may cause depressive symptoms such as anhedonia. Using our SPT protocol, more elaborate rodent models can be developed to study the mechanisms underlying inflammation-associated depression in humans.

Systemic inflammation in early neonatal mice induces transient and lasting neurodegenerative effects.

BackgroundThe inflammatory mediator lipopolysaccharide (LPS) has been shown to induce acute gliosis in neonatal mice. However, the progressive effects on the murine neurodevelopmental program over the week that follows systemic inflammation are not known. Thus, we investigated the effects of repeated LPS administration in the first postnatal week in mice, a condition mimicking sepsis in late preterm infants, on the developing central nervous system (CNS).MethodsSystemic inflammation was induced by daily intraperitoneal administration (i.p.) of LPS (6 mg/kg) in newborn mice from postnatal day (PND) 4 to PND6. The effects on neurodevelopment were examined by staining the white matter and neurons with Luxol Fast Blue and Cresyl Violet, respectively. The inflammatory response was assessed by quantifying the expression/activity of matrix metalloproteinases (MMP), toll-like receptor (TLR)-4, high mobility group box (HMGB)-1, and autotaxin (ATX). In addition, B6 CX3CR1gfp/+ mice combined with cryo-immunofluorescence were used to determine the acute, delayed, and lasting effects on myelination, microglia, and astrocytes.ResultsLPS administration led to acute body and brain weight loss as well as overt structural changes in the brain such as cerebellar hypoplasia, neuronal loss/shrinkage, and delayed myelination. The impaired myelination was associated with alterations in the proliferation and differentiation of NG2 progenitor cells early after LPS administration, rather than with excessive phagocytosis by CNS myeloid cells. In addition to disruptions in brain architecture, a robust inflammatory response to LPS was observed. Quantification of inflammatory biomarkers revealed decreased expression of ATX with concurrent increases in HMGB1, TLR-4, and MMP-9 expression levels. Acute astrogliosis (GFAP+ cells) in the brain parenchyma and at the microvasculature interface together with parenchymal microgliosis (CX3CR1+ cells) were also observed. These changes preceded the migration/proliferation of CX3CR1+ cells around the vessels at later time points and the subsequent loss of GFAP+ astrocytes.ConclusionCollectively, our study has uncovered a complex innate inflammatory reaction and associated structural changes in the brains of neonatal mice challenged peripherally with LPS. These findings may explain some of the neurobehavioral abnormalities that develop following neonatal sepsis.

Etazolate abrogates the lipopolysaccharide (LPS)-induced downregulation of the cAMP/pCREB/BDNF signaling, neuroinflammatory response and depressive-like behavior in mice

Increasing evidence has indicated that immune challenge by bacterial lipopolysaccharide (LPS) induces depressive-like behavior, neuroinflammatory response and upregulates phosphodiesterase-4 (PDE4), an enzyme that specifically hydrolyzes cyclic adenosine monophosphate (cAMP). However, whether the potential PDE4 inhibitor etazolate prevents the LPS-induced depressive-like behavior remains unclear. Here using a model of depression induced by the repeated administration of LPS during 16days, and then investigated the influence of LPS on the expression of PDE4, interleukin-1β (IL-1β) and antidepressant action of etazolate in mice through forced swimming, novelty suppressed feeding, sucrose preference and open-field tests. Our results showed that etazolate pretreatment facilitated the recovery from weight loss and prevented the depressive-like behavior induced by repeated LPS administration. Moreover, the antidepressant action of etazolate was paralleled by significantly reducing the expression levels of PDE4A, PDE4B, PDE4D and IL-1β and up-regulating the cAMP/phosphorylated cAMP response-element binding protein (pCREB)/brain-derived neurotrophic factor (BDNF) signaling in the hippocampus and prefrontal cortex of mice. These results indicate that the effects of etazolate on the depressive-like behavior induced by repeated LPS treatment may partially depend on the inhibition of PDE4 subtypes, the activation of the cAMP/pCREB/BDNF signaling and the anti-inflammatory responses in the hippocampus and prefrontal cortex.

Species-Dependent Blood-Brain Barrier Disruption of Lipopolysaccharide: Amelioration by ColistinIn VitroandIn Vivo

The aim of this study was to use in vitro and in vivo models to assess the impact of lipopolysaccharide (LPS) from two different bacterial species on blood-brain barrier (BBB) integrity and brain uptake of colistin. Following repeated administration of LPS from Pseudomonas aeruginosa, the brain-to-plasma ratio of [14C]sucrose in Swiss outbred mice was not significantly increased. Furthermore, while the brain uptake of colistin in mice increased 3-fold following administration of LPS from Salmonella enterica, LPS from P. aeruginosa had no significant effect on colistin brain uptake. This apparent species-dependent effect did not appear to correlate with differences in plasma cytokine levels, as the concentrations of tumor necrosis factor alpha and interleukin-6 following administration of each LPS were not different (P > 0.05). To clarify whether this species-specific effect of LPS was due to direct effects on the BBB, human brain capillary endothelial (hCMEC/D3) cells were treated with LPS from P. aeruginosa or S. enterica and claudin-5 expression was measured by Western blotting. S. enterica LPS significantly (P < 0.05) reduced claudin-5 expression at a concentration of 7.5 μg/ml. In contrast, P. aeruginosa LPS decreased (P < 0.05) claudin-5 expression only at the highest concentration tested (i.e., 30 μg/ml). Coadministration of therapeutic concentrations of colistin ameliorated the S. enterica LPS-induced reduction in claudin-5 expression in hCMEC/D3 cells and the perturbation in BBB function in mice. This study demonstrates that BBB disruption induced by LPS is species dependent, at least between P. aeruginosa and S. enterica, and can be ameliorated by colistin.

Electrical activity in rat cortico-limbic structures after single or repeated administration of lipopolysaccharide or staphylococcal enterotoxin B

Immune-to-brain communication is essential for an individual to aptly respond to challenging internal and external environments. However, the specificity by which the central nervous system detects or 'senses' peripheral immune challenges is still poorly understood. In contrast to post-mortem c-Fos mapping, we recorded neural activity in vivo in two specific cortico-limbic regions relevant for processing visceral inputs and associating it with other sensory signalling, the amygdala (Am) and the insular cortex (IC). Adult rats were implanted with deep-brain monopolar electrodes and electrical activity was monitored unilaterally before and after administration of two different immunogens, the T-cell-independent antigen lipopolysaccharide (LPS) or the T-cell-dependent antigen staphylococcal enterotoxin B (SEB). In addition, the neural activity of the same individuals was analysed after single as well as repeated antigen administration, the latter inducing attenuation of the immune response. Body temperature and circulating cytokine levels confirmed the biological activity of the antigens and the success of immunization and desensitization protocols. More importantly, the present data demonstrate that neural activity of the Am and IC is not only specific for the type of immune challenge (LPS versus SEB) but seems to be also sensitive to the different immune state (naive versus desensitization). This indicates that the forebrain expresses specific patterns of electrical activity related to the type of peripheral immune activation as well as to the intensity of the stimulation, substantiating associative learning paradigms employing antigens as unconditioned stimuli. Overall, our data support the view of an intensive immune-to-brain communication, which may have evolved to achieve the complex energetic balance necessary for mounting effective immunity and improved individual adaptability by cognitive functions.

Reproductive axis response to repeated lipopolysaccharide administration in peripubertal female rats

Immune system disorders are often accompanied by alterations in the reproductive axis. Several reports have shown that administration of bacterial lipopolysaccharide (LPS) has central inflammatory effects and activates cytokine release in the hypothalamus where the luteinizing hormone releasing hormone (Gn-RH) neurons are located. The present study was designed to investigate the effect of repeated LPS administration on the neuroendocrine mechanisms of control of the reproductive axis in peripubertal female rats (30-day-old rats). With this aim, LPS (50 mug/kg weight) was administered to the animals during 25, 27 and 29 days of age and sacrificed on 30 day of life. Gn-RH, gamma-amino butyric acid (GABA) and glutamic acid (GLU), two amino acids involved in the regulation of Gn-RH secretion, hypothalamic content were measured. LH and estradiol serum levels were also determined and the day of vaginal opening examined. The results showed a significant increase in Gn-RH and GLU content (p < 0.0001), shared by a reduction of GABA one (p < 0.0001). LH and estradiol serum levels were decreased (p < 0.01, p < 0.001) and delay in the day of vaginal opening was also observed in treated animals. Present results show that repeated LPS administration impaired reproductive function, modifying the neuroendocrine mechanisms of control of the axis in peripubertal female rats.

Differential sensitivity to endotoxin exposure in young and middle-age mice

Aging can have a profound effect on the neurobehavioral response to immune activation; aged subjects are predisposed to greater deficits in performance and cognitive function in conjunction with an exaggerated neuroinflammatory response. While increased reactivity to an immune insult has been well characterized in aged subjects, the alterations that may exist by middle-age have not been thoroughly investigated. The present study compared the reactions of young (4-month) and middle-age (12-month) male BALB/c mice to an acute or repeated lipopolysaccharide (LPS) challenge(s). The data suggest that in some respects middle-aged mice are more sensitive to endotoxin exposure, as they show enhanced weight loss, splenic cytokine levels, and c-fos expression in the brain following acute LPS administration compared to younger mice. However, acute LPS exposure led to comparable decreases in locomotor activity in young and middle-aged mice. Following repeated LPS administration both age groups showed diminished behavioral and neural reactions to the final LPS challenge, indicating tolerance development. However, the immune system of the middle-aged mice was still mildly responsive to the final LPS exposure, as splenic levels of IL-1β were significantly elevated. Collectively, the data suggest that middle-age subjects are more sensitive to an immune insult.

Locally administered T cells from mice immunized with lipopolysaccharide (LPS) accelerate LPS-induced bone resorption

T cells play important roles in bone destruction and osteoclastogenesis and are found in chronic destructive bone lesions. Lipopolysaccharide (LPS) is one of several pathological factors involved in inflammatory bone destruction. We previously described the importance of T cells in the inflammatory bone resorption that occurs after repeated LPS administration. However, whether local or systemic T cells are important for inflammatory bone resorption and whether immunization of host animals influences bone resorption remain unclear. The present study examines the effects of local extant T cells from LPS-immunized mice on LPS-induced bone resorption. T cells from LPS-immunized or non-immunized mice were injected together with LPS into the gingival tissues of mice with severe combined immunodeficiency disease that lack both T and B cells. We histomorphometrically evaluated bone resorption at sites of T cell injections and examined the influence of T cells from LPS-immunized mice on osteoclastogenesis in vitro. We found that locally administered T cells from LPS-immunized but not non-immunized mice accelerated LPS-induced bone resorption in vivo. Moreover, T cells from LPS-immunized mice increased osteoclastogenesis in vitro induced by receptor activator of NF-κ B ligand and LPS and anti-tumor necrosis factor (TNF)-α antibody inhibited this increase. These results demonstrated that local extant T cells accelerate inflammatory bone resorption. Furthermore, T cells from LPS-immunized mice appear to elevate LPS-induced bone resorption using TNF-α.

S1731 Tolerance of Toll-Like Receptor 4 Mediated Signaling Pathway Protects Mice from Experimental Colitis

Background:The basis of inflammatory bowel disease (IBD) is multi-factorial involving susceptibility genes, and immune and environmental factors. There has been a rapid increase in the prevalence of IBD in industrialized countries. The hygiene hypothesis proposes that the present clean surroundings in less exposure to bacteria are involved in development of this disease. Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), has a phenomenon termed LPS tolerance such as an initial exposure to LPS induce a transient state of hyporesponsiveness to a subsequent challenge with LPS. Based on In Vitro studies, the mechanisms of this phenomenon are gradually becoming clear, although that has not been formally proven in inflammatory disease models In Vivo. Therefore we hypothesized that repeated LPS administration could protect colonic inflammation of mouse experimental colitis model. Methods:Murine colitis was induced to Balb/c mice by oral administration of 7% dextran sulphate sodium (DSS) with or without daily intraperitoneal administration of LPS. Colitis was evaluated by a macroscopic disease activity index, myeloperoxidase (MPO) activity in colon and histological score. Cytokine mRNA expressions in colon were also measured by RT-PCR. To confirm the phenomenon of LPS tolerance, we generated mouse conventional bone marrow derived dendritic cells (BMDC), and these were preincubated with or without LPS and restimulated with LPS after 24 hours from first stimulation. Cytokine productions in the culture supernatant were measured by ELISA, and mRNA expressions of the cells were evaluated by RT-PCR. Furthermore, we investigated negative regulators of LPS tolerance and expression of them in model of experimental colitis. Results:Administration of LPS significantly suppressed colonic inflammation of DSS-induced colitis, although there were no difference in mRNA expression of IL-12p40, IL-6, TNF-α, IL-10, and TLR4. TLR4 in BMDC was activated with LPS and induced TNF-α production. After second stimulation with LPS, TNF-α and IL-6 production were reduced which followed previous reports. IRAKM mRNA expression, which had participation in LPS tolerance, was upregulated in the LPS treated BMDC and mice colon in comparison to the control mice. Conclusion:LPS tolerance could protect mice from DSS-induced colitis, and IRAK-M had participation in LPS tolerance of TLR4 signaling pathway. Taken together, these observations suggested that loss of exposure to LPS may be one of the pathogenesis of IBD.

The influence of age and repeated lipopolysaccharide administration on body temperature and the concentration of interleukin-6 and IgM antibodies against lipopolysaccharide in broiler chickens

Our objective was to create a standardized and reproducible inflammation model in chickens in order to study the pharmacodynamics of several anti-pyretic and anti-inflammatory drugs. We studied the influence of age and repeated lipopolysaccharide (LPS) administration on body temperature and the correlation of this with concentrations of interleukin-6 and IgM antibodies against LPS in plasma of chickens. Three-week-old and 5-week-old broilers were injected intravenously with LPS from Escherichia coli O127: B8 at a dose of 1 mg/kg. LPS administration was repeated after 2 or 7 days. After the first dose of LPS, the body temperature was initially decreased below normal and then later increased above normal. The second dose of LPS reduced the level of hypothermia and the duration of the febrile phase. Three-week-old birds responded to LPS with a higher maximum body temperature and a greater area under the body temperature versus time curve than 5-week-old chickens (P<0.05). Interleukin-6 reached its highest concentration 3 h after LPS administration and returned to baseline levels after 9 h. A second dose of LPS resulted in a significantly lower peak in interleukin-6. Significant higher levels of antibodies against LPS could be detected 7 days after LPS administration. However, there appeared to be no correlation between the reduced response to LPS and the presence of antibodies.

Weaken taste-LPS association during endotoxin tolerance

In naive individuals, the administration of bacterial lipopolysaccharide (LPS) provokes a rapid systemic increase in pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, inducing an acute phase response including sickness behavior. Strong associative learning occurs when relevant gustatory/olfactory stimuli precede the activation of the immune system, affecting long-term individual food selection and nutritional strategies. Repeated LPS administration results in the development of an endotoxin tolerance status, characterized by a drastic reduction in the LPS-induced cytokine response. Here we investigated how the postprandial categorization of a relevant taste (0.2% saccharin) changed after administration of a high dose of LPS (0.5 mg/kg i.p.) in LPS-tolerant animals. Determination of the consummatory fluid intake revealed that, in contrast to LPS-naive rats, taste-LPS association did not occur during endotoxin tolerance. Ninety minutes after the single association trial, the plasma responses of TNF-α, IL-1β and IL-6 were completely blunted in LPS-tolerant animals, which also resulted in low LPS-adipsogenic and LPS-anorexic effects. These findings indicate that an identical immune challenge can result in completely different neuro-behavioral consequences depending on the immune history of the individual, thus revealing part of the complex interconnection between the immune and neuro-endocrine systems in regulating food selection and consumption during the infectious process.

Expression of hypothalamic neuropeptides and the desensitization of pituitary–adrenal axis and hypophagia in the endotoxin tolerance

Repeated exposure to lipopolysaccharide (LPS) induces desensitization of hypothalamus–pituitary–adrenal axis (HPA) responses and hypophagia. We investigated the interplay between the neural circuitries involved in the control of food intake and HPA axis activity following single or repeated LPS injections. Male Wistar rats received a single or repeated i.p. injection of LPS (100 μg/kg) for 6 days and were subdivided into four groups: 6 saline, 5 saline + 1 LPS, 5 LPS + 1 saline and 6 LPS. Animals with a single exposure to LPS showed increased plasma levels of ACTH, CORT, PRL, TNF-α and also CRF mRNA in the paraventricular nucleus of the hypothalamus. These animals exhibited a reduced food intake and body weight associated with an increase of CART expression in the arcuate nucleus (ARC). Leptin plasma levels were not altered. On the other hand, repeated LPS administration did not alter ACTH, CORT, PRL and TNF-α, but it reduced leptin level, compared to single LPS or saline treatment. Furthermore, repeated LPS administration did not increase CRF or CART mRNA expression. Food intake and weight gain after repeated LPS injections were not different from saline-treated animals. There was no change in NPY and POMC mRNA expression in the ARC after single or repeated injections of LPS. In conclusion, desensitization induced by repeated exposure to LPS involves the blockade of HPA axis activation and anorexigenic response, which are both associated with an unresponsiveness of TNF-α production and CRF and CART expression in the hypothalamus.