Post Lipopolysaccharide Challenge Research Articles

Identification of novel biomarkers of acute phase response in chickens challenged with Escherichia coli lipopolysaccharide endotoxin

BackgroundThe chicken’s inflammatory response is an essential part of the bird’s response to infection. A single dose of Escherichia coli (E. coli) lipopolysaccharide (LPS) endotoxin can activate the acute phase response (APR) and lead to the production of acute phase proteins (APPs). In this study, the responses of established chicken APPs, Serum amyloid A (SAA) and Alpha-1-acid-glycoprotein (AGP), were compared to two novel APPs, Hemopexin (Hpx) and Extracellular fatty acid binding protein (Ex-FABP), in 15-day old broilers over a time course of 48 h post E.coli LPS challenge. We aimed to investigate and validate their role as biomarkers of an APR. Novel plant extracts, Citrus (CTS) and cucumber (CMB), were used as dietary supplements to investigate their ability to reduce the inflammatory response initiated by the endotoxin.ResultsA significant increase of established (SAA, AGP) and novel (Ex-FABP, Hpx) APPs was detected post E.coli LPS challenge. Extracellular fatty acid binding protein (Ex-FABP) showed a similar early response to SAA post LPS challenge by increasing ~ 20-fold at 12 h post challenge (P < 0.001). Hemopexin (Hpx) showed a later response by increasing ∼5-fold at 24 h post challenge (P < 0.001) with a similar trend to AGP. No differences in APP responses were identified between diets (CTS and CMB) using any of the established or novel biomarkers.ConclusionsHpx and Ex-FABP were confirmed as potential biomarkers of APR in broilers when using an E. coli LPS model along with SAA and AGP. However, no clear advantage for using either of dietary supplements to modulate the APR was identified at the dosage used.

237 Disrupted insulin signaling due to an acute immune response in swine

Abstract Insulin is an anabolic hormone involved in glucose uptake and synthesis of fats, proteins, and glycogen. Altered insulin signaling can occur due to metabolic state, pathogen exposure, and autoimmune disorders. Lipopolysaccharide (LPS), an endotoxin secreted produced by gram-negative bacteria, can induce a severe, systemic immune response. In pigs, chronic LPS exposure has induced insulin resistance. However, the effects of acute exposure to LPS on insulin signaling and resistance have not been elucidated. Crossbred nursery pigs [n = 58, initial body weight (BW) = 12.1 kg ± 0.23 kg] were randomly assigned into two experimental subsets, where 10 animals were designated for repeated blood sampling and 48 animals underwent timed euthanasia for tissue collection. For repeated blood sampling, jugular catheters were placed under anesthesia and the animals were given 48 h to recover before being randomly assigned to treatments. These treatments were an intraperitoneal injection of either 15µg/kg BW LPS or an equal volume of 0.9% Saline (n = 5 pigs/treatment). Blood was collected for 48 h followed by euthanasia. The other 48 pigs were randomly assigned to the same treatments, and then randomly assigned to euthanasia timepoints of 0, 3, 6, 12, 24 or 48 h post injection for tissue collection. Among the catheterized pigs, 2-h post LPS challenge there was a decrease in serum insulin concentration (P &amp;lt; 0.05) and mild hyperglycemia (P &amp;lt; 0.05) post challenge. At 12- and 24-h post LPS challenge there was a decrease in insulin (P &amp;lt; 0.05) and a trend hyperglycemia was also apparent at 24 h (P = 0.063). In liver tissue, insulin related genes exhibited alterations post LPS injection. Phosphoinositide 3-kinase (pi3k) and insulin receptor substrate 1 (irs1) expression were decreased at 3 and 6 h (p&amp;lt; 0.05) and protein kinase b (akt) expression were decreased at 3 h (P &amp;lt; 0.05) post challenge. The expression of insulin inhibitor growth factor receptor bound protein 10 (grb10) was decreased at 6-, 12-, and 24-h after challenge (P &amp;lt; 0.05). Fructose-1-6-bisphosphatase (f16bp) expression was reduced at 3-, 6-, and 12-h (P &amp;lt; 0.05) in the liver while phosphoenolpyruvate carboxykinase (pepck) expression was decreased at 3- and 6-h (P &amp;lt; 0.01) post challenge. Other metabolic genes were decreased at 3-, 6-, and 12-h post LPS challenge, including fatty acid synthase (fas; P &amp;lt; 0.05), carbohydrate response element binding protein (chrebp; P &amp;lt; 0.05), and acetyl CoA carboxylase (acc; P &amp;lt; 0.05). Protein abundance of Akt 3 h after LPS injection was decreased in the liver (P &amp;lt; 0.05). In skeletal muscle 6 h post LPS injection, insulin receptor (insr), insulin-like growth factor receptor (igfr) and fas were all increased (P &amp;lt; 0.05) These results indicate disrupted insulin signaling at various levels after an acute immune response.

147 The interaction between growth and immunity: Insights into the immune response of finisher pigs with divergent feed efficiency

Abstract This work evaluated the immune response of finisher pigs with divergent feed efficiency to identify immune pathways that were associated with efficiency. Blood gene expression data from a previous trial (Liu et al., 2019) were re-analyzed with novel methods that quantified changes in biological pathways and associated them with animal performance. Data were quality control checked and aligned to the Sus scrofa reference genome (Sscrofa11.1) using Kallisto. The dataset had 32 samples from eight pigs with high efficiency (HE) or low efficiency (LE) that had been injected with lipopolysaccharide (LPS). Blood samples were taken before challenge, and at 2, 6, and 24 h after challenge. After normalization and evaluation of differential gene expression, activation or inhibition of pathways was quantified using a topology-based pathway activation method and a customization of the Reactome database. Pathways were associated with feed efficiency using a regularized regression algorithm to identify immune pathways that were associated with efficiency. For statistical analysis, RNA integrity number, feed efficiency and time post LPS challenge were included in the model. At 2 h post LPS, HE pigs showed greater activation in pathways related to heat stress and creatine metabolism, but inhibition of pathways related to lysine and threonine catabolism, and immune resolving mediators (P-adj &amp;lt; 0.01), compared with the response in LE pigs. At 6 h post challenge there was a striking difference in cell cycle response, whereby cell cycle was activated in HE pigs but inhibited in LE pigs (P-adj &amp;lt; 0.01). The biggest difference in immune response was at 24h, with a strong response to LPS still evident in HE pigs, but a diminishing response in LE pigs. High efficiency pigs had activation in pathways related to oxidative stress, the metabolism of tryptophan, serine, tyrosine, threonine, glycogen, vitamin B5, and fibrin clot formation (P-adj &amp;lt; 0.01), which were not evident in LE pigs. The regression of pathways with feed efficiency showed that vitamin B5 metabolism was positively associated with gain:feed, whilst calcium-dependent signaling was negatively associated with gain:feed at multiple timepoints post-challenge. High efficiency pigs appeared to expand leukocyte populations at 6 h post LPS challenge, which was not evident in LE pigs. By 24 h, the response of LE pigs to LPS was minimal, but HE pigs still showed a strong response with greater utilization of energy, amino acids, and more oxidative stress. Low efficiency pigs appeared to recover faster from the LPS challenge, which was partially explained by differences in vitamin B5 utilization and calcium homeostasis. These findings confirm differences in the immune response of HE and LE pigs and provide insights related to amino acid, vitamin B5, and calcium homeostasis, which could help optimize the immune response of HE pigs. The novel analysis methods provided additional insights, which complement the original findings (T1).

Ciona robusta macrophage migration inhibitory factor (Mif1 and Mif2) genes are differentially regulated in the LPS-challenged pharynx.

The effects of lipopolysaccharide (LPS) on Mif (macrophage migration inhibitory factor) gene expression in the pharynx (hemapoetic tissue) of Ciona robusta was investigated using qRT-PCR, and ISH (in situ hybridisation). To verify the induction of an inflammatory response in the pharynx, a qRT-PCR analysis was performed to evaluate the change in expression of proinflammatory marker genes such as Mbl, Ptx-like, Tnf-α and Nf-kb, which were shown to be upregulated at 1 hour post LPS challenge. The change in expression of the two Mif paralogs in the pharynx were assessed prior to and following stimulation and qRT-PCR, and ISH results showed that, while Mif2 and Mif2 were expressed in clusters of hemocytes in pharynx vessels, only Mif1 expression increased following LPS stimulation. This indicates that the Mif genes are differently regulated and respond to different ambient inputs that are in need of further analysis. This article is protected by copyright. All rights reserved.

RNA form baker's yeast cultured with and without lipopolysaccharide (LPS) modulates gene transcription in an intestinal epithelial cell model, RTgutGC from rainbow trout (Oncorhynchus mykiss)

The objective of this study was to evaluate if the intestinal RTgutGC cell line could be suitable for research on dietary ingredients and their function as modulators of inflammation during lipopolysaccharide (LPS) induced stress.The RTgutGC cells cultured together with RNA from baker's yeast, reached confluency after 72 h. The cells were grown in either compete L-15 (CM) or nutrient deprived L-15 (DM). Then, the RTgutGC cells were exposed to LPS or RNA from baker's yeast, either alone, or in combination, in CM or DM. All cultures were harvested following LPS challenge for 48 h and 72 h.LPS induced transcription of Interleukin 1β (IL-1β), Interleukin −8 (IL-8), Toll like receptor 3 (TLR3), interferon regulating factor 3 (irf3), Nuclear factor ĸβ (NFĸβ), one of the multidrug transporters, ABCC2, and glutamine synthase 1 (GLS01) in RTgutGC cells at one or both sampling points (48 h and/or 72 h post LPS challenge). RNA from baker's yeast in culture alone, (cultured 120 h and 144 h with RTgutGC cells and harvested at the respective LPS sampling points) induced transcription of INF1, TNFα and ticam/trif, not induced by LPS. In addition, RNA from baker's yeast affected IL-1β, TLR3, irf3 and NFĸβ, comparable to the responses triggered by LPS. RNA from baker's yeast alone did not affect ABCC2 or GLS01 transcriptions in this set up. So, LPS and RNA from baker's yeast affects distinct but also common gene transcripts in this intestinal cell line.Culturing RTgutGC cells in DM, adding a combination of LPS and RNA from baker's yeast, reduced IL-1β transcription compared to cells grown in CM, 48 h and 72 h post LPS challenge. Also, in RTgutGC cells, grown in DM, the LPS induced transcription of ABCC2 declined, measured 48 h post LPS challenge. Possibly indicating that optimal transcription of IL-1β and ABBC2 in RTgutGC cells, cultured over time, requires access of adequate nutrients under stressful condition.RNA from baker's yeast induced INF1 transcription in the RTgutGC cells, regardless if the medium was complete or deprived of nutrients. However, culturing RTgutGC cells in DM enriched with RNA from baker's yeast for a longer period of time (120 h, 144 h), seemed beneficial for INF1 transcription.

Transcriptome profiling reveals new insights into the roles of neuronal nitric oxide synthase on macrophage polarization towards classically activated phenotype.

In response to various stimuli, naïve macrophages usually polarize to M1 (classically activated) or M2 (alternatively activated) cells with distinct biological functions. Neuronal nitric oxide synthase (NOS1) is involved in M1 macrophage polarization at an early stage. Here, we show for the first time that NOS1 is dispensable for M2 macrophage polarization for the first time. Further, differentially expressed genes (DEGs) regulated by NOS1 signaling in M1-polarized macrophages stimulated with lipopolysaccharide (LPS) were characterized by transcriptome analysis of wild-type (WT) and NOS1 knockout mouse macrophages. Thousands of affected genes were detected 2 h post LPS challenge, and this wide-ranging effect became greater with a longer stimulation time (8 h post LPS). NOS1 deficiency caused dysregulated expression of hundreds of LPS-responsive genes. Most DEGs were enriched in biological processes related to transcription and regulation of the immune and inflammatory response. At 2 h post-LPS, the toll-like receptor (TLR) signaling pathway, cytokine–cytokine receptor interaction, and NOD-like receptor signaling pathway were the major pathways affected, whereas the main pathways affected at 8 h post-LPS were Th1 and Th2 cell differentiation, FoxO, and AMPK signaling pathway. Identified DEGs were validated by real-time quantitative PCR and interacted in a complicated signaling pathway network. Collectively, our data show that NOS1 is dispensable for M2 macrophage polarization and reveal novel insights in the role of NOS1 signaling at different stages of M1 macrophage polarization through distinct TLR4 plasma membrane-localized and endosome-internalized signaling pathways.

Effect of nanocurcumin and fish oil as natural anti-inflammatory compounds vs. glucocorticoids in a lipopolysaccharide inflammation model on Holstein calves’ health status

Curcumin (CUR) and fish oil (FO) are among the most well-known types of natural anti-inflammatory compounds. The aim of this study was to assess the effect of nanocurcumin and fish oil vs. glucocorticoids on Holstein calves’ health status. A lipopolysaccharide (LPS) challenge (0.5 μg kg−1 BW) was used to induce an acute phase response. A total of 42 male Holstein calves were randomized into 7 groups: negative control (CON), positive control (LPS, injected once), 250 mg/kg BW per day fish oil + LPS (FO250), 350 mg/kg BW per day fish oil + LPS (FO350), 2 mg/kg BW per day nanocurcumin + LPS (NCUR2), 4 mg/kg BW per day nanocurcumin + LPS (NCUR4), and 0.3 mg/kg BW dexamethasone (injected once) + LPS (DEX). The duration of this experiment was 11 days, with application of the LPS challenge on day 8. Calves were weighed on days 0, 7, 9, 10, and 11 to record the average daily weight gain; diets offered and refused were recorded daily throughout the experiment. Blood collection and clinical scoring were conducted at successive time points until 72 h post LPS challenge. The data obtained also comprised rectal temperature (RT), respiratory rate (RR), heart rate (HR), concentrations of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), serum amyloid A (SAA), and haptoglobin (Hp). This experiment could not uncover significant effects of LPS, FO, NCUR, and DEX on the area under the curve (AUC) of the RT, HR, and RR; in addition, there was no difference between FO and NCUR vs. LPS in sickness behavior, however, DEX group significantly recovered faster than others (P < 0.01). There was no significant difference between groups in dry matter intake (DMI) and average daily gain (ADG) during three days post LPS challenge. The concentrations of TNF-⍺, IL-6, and SAA were lower in the DEX group (P < 0.05). Finally, no effects of FO and NCUR on cytokines and acute phase proteins (APPs) could be observed in this study. In conclusion, supplementation of FO and NCUR was not able to impact the acute phase response (APR) in calves, as levels of inflammatory cytokines and APPs as well as sickness behavior remained unchanged. It seems that the anti-inflammatory effects of FO and CUR on APR, as has been observed for other animal species, do not manifest that clearly in calves.

Glycine Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Regulating NLRP3 Inflammasome and NRF2 Signaling

Glycine supplementation has been reported to alleviate lipopolysaccharide (LPS)-induced lung injury in mice. However, the underlying mechanisms responsible for this beneficial effect remain unknown. In the present study, male C57BL/6 mice were treated with aerosolized glycine (1000 mg in 5 mL of 0.9% saline) or vehicle (0.9% saline) once daily for 7 continuous days, and then were exposed to aerosolized LPS (5 mg in 5 mL of 0.9% saline) for 30 min to induce lung injury. Sera and lung tissues were collected 24 h post LPS challenge. Results showed that glycine pretreatment attenuated LPS-induced decreases of mucin at both protein and mRNA levels, reduced LPS-triggered upregulation of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interferons, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukins. Further study showed that glycine-reduced LPS challenge resulted in the upregulation of nuclear factor κB (NF-κB), nucleotide binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome. In addition, LPS exposure led to the downregulation of NRF2 and downstream targets, which were significantly improved by glycine administration in the lung tissues. Our findings indicated that glycine pretreatment prevented LPS-induced lung injury by regulating both NLRP3 inflammasome and NRF2 signaling.

185 Impact of hydroxy-selenomethionine supplementation during late gestation and lactation in sows on piglet immunocompetence

Abstract The benefits of selenium (Se) supplementation during pregnancy and lactation in sows on piglet immunocompetence are not yet fully understood. This study aimed to assess piglet immune status when sows were fed Se-deficient or supplemented diets with sodium selenite (SS) or hydroxy-selenomethionine (OH-SeMet). Twelve pregnant sows were randomly assigned to 3 dietary treatments (4 sows each): no supplemental Se (NC), SS at 0.3 mg Se/kg (SS-0.3), OH-SeMet at 0.3 mg Se/kg (SO-0.3). The trial was carried out from gestation day (gd) 84 to weaning on postpartum day (ppd) 21, at which time all the piglets received the same diet having as Se source SS. On gd 98 and 105, sows were vaccinated with hen egg white lysozyme (HEWL). On ppd 0 and 21, 8 piglets/treatment were euthanized to obtain blood and muscle samples for total Se analyses. Serum samples were collected from 8 female piglets/treatment on ppd 0 and 2 to assess antigen-specific antibody titer for HEWL. On ppd 23, 6 weaned female piglets/treatment were intramuscular challenged with E. coli lipopolysaccharide (LPS) and serum IL-10 levels were measured at 24 h pre-challenge and at 2 and 48 h post challenge. At day 21, piglets from sows fed SO-0.3 showed the highest muscle Se deposition (P &lt; 0.001). At ppd 0, no difference for HEWL-specific IgG was found while at ppd 2, SO-0.3 showed the highest HEWL-specific IgG concentration (P &lt; 0.05). At 24 h pre-challenge and 2 h post LPS challenge, SO-0.3 showed a greater IL-10 level as compare to NC and SS-0.3, respectively (P &lt; 0.05); no difference was found for the same parameter at 48 h post challenge. Piglets from SO supplemented sows exhibited stronger early immune response to LPS challenge. This study indicates the importance of sow Se supplementation with a pure organic form of Se such as OH-SeMet to enhance piglets Se and immune status.

Astragalin reduces lipopolysaccharide-induced acute lung injury in rats via induction of heme oxygenase-1.

Astragalin, a bioactive component of medicinal plants such as Rosa agrestis, has anti-inflammatory and antioxidant features. Induction of heme oxygenase (HO)-1 is an effective strategy to reduce excessive generated oxidants during the pathogenesis of acute lung injury (ALI). The aim of the present study is to investigate that whether the anti-inflammatory and antioxidant features of astragalin is HO-1 dependent in lipopolysaccharide (LPS)-induced ALI. Sprague-Dawley rats were used in animal study. Intratracheal LPS was performed to induce experimental ALI model. Astragalin was administrated 1h after LPS challenge. Human lung epithelial cells were used in cell study. Samples from rats were harvested at 24h post LPS challenge. Astragalin treatment inhibited LPS-induced inflammatory cells infiltration in the lung and pulmonary edema. Astragalin treatment markedly enhanced the activity of HO-1 compared with vehicle-treated group at 24h post LPS challenge. Levels of lipid hydroperoxide, a marker for oxidative stress, were decreased in astragalin-treated animals compared with vehicle-treated group. However, the protective effect of astragalin on LPS-induced ALI was abolished in an inhibitor of HO-1-treated animals. Moreover, the astragalin-induced the upregulation of HO-1 in human lung epithelial cells was inhibited when nuclear factor erythroid-2-related factor 2 (Nrf2) was silenced by small interfering RNA. Astragalin reduces LPS-induced ALI via activation of Nrf2/HO-1 pathway.

Anti-inflammatory mechanism of ginsenoside Rg1: Proteomic analysis of milk from goats with mastitis induced with lipopolysaccharide

Previous investigation showed that intravenous injection of ginsenoside Rg1 had a therapeutic effect on Escherichia coli lipopolysaccharide-induced mastitis in lactating goats and it protected animals from lipopolysaccharide challenge via toll-like receptor 4 signaling pathway. The present study was to use proteomic approach to explore the anti-inflammatory mechanisms of Rg1. Nine dairy goats were randomly divided into three groups with 3 animals in each: groups 1 and 2 received intra-mammary infusion of lipopolysaccharide and then intravenously injected with saline or Rg1 solution; animals in group 3 were first intramammarily and then intravenously administered saline solution, and served as a control group. Milk whey at 6 h post lipopolysaccharide challenge was prepared for tandem mass tags based quantitative proteomic analysis. The results showed that 791 proteins were totally identified from the whey. Of them, 98 proteins between groups 1 (lipopolysaccharide + Saline) and 3 (Saline + Saline), and 34 proteins between groups 2 (lipopolysaccharide + Rg1) and 1 were significantly different. Group 1 than group 3 had significantly more inflammatory factors such as interleukin 6, acute phase proteins, blood coagulation factors, complement proteins, and oxidative stress markers while these factors were reduced in group 2 treated with Rg1. In addition, proteins in group 2 associated with peroxisome-proliferator-activated receptor γ activation and recovery of milk fat and production were upregulated compared to group 1. Therefore, Rg1 may exert its anti-inflammatory effect on lipopolysaccharide-induced mastitis in goats via modulating expression of proteins relating to peroxisome-proliferator-activated receptor γ and toll-like receptor 4 signaling pathway.

Therapeutic effect of ginsenoside Rg1 on mastitis experimentally induced by lipopolysaccharide in lactating goats

Escherichia coli is a cause of subclinical and clinical mastitis in dairy cattle and goats, and sometimes causes severe clinical disease that may result in death of the animal. Previous investigation showed that ginsenoside Rg1 extracted from Panax ginseng C.A. Meyer (Araliaceae) has an anti-inflammatory effect on the sepsis induced by E. coli lipopolysaccharide via competitive binding to toll-like receptor 4. We hypothesized that intravenous injection of Rg1 had therapeutic effect on mastitis experimentally induced by intramammary infusion of lipopolysaccharide in lactating goats. In this study, 9 lactating goats were randomly assigned to 1 of the 3 groups: (1) lipopolysaccharide intramammary infusion + saline intravenous injection, (2) lipopolysaccharide intramammary infusion + Rg1 intravenous injection, and (3) saline intramammary administration + saline intravenous injection. Because no adverse clinical signs were observed after intramammary infusion of saline and intravenous injection of Rg1 in a preliminary experiment, and available qualified goats were limited in this study, this treatment was not included in this study. One udder half of each goat received intramammary infusion of lipopolysaccharide (50 μg/kg of body weight; groups 1 and 2) or saline solution (group 3), and the other half was infused with 2 mL of saline solution at h 0. Afterward, intravenous injections of saline solution (groups 1 and 3) or Rg1 (2.5 mg/kg of body weight; group 2) were administered at h 2 and 4 post-lipopolysaccharide challenge. Blood and milk samples were collected 3, 6, 9, 12, 15, 18, 21, 24, 48, and 72 h post-lipopolysaccharide challenge, and clinical signs were monitored hourly after lipopolysaccharide challenge within the first 10 h and at the same time points as blood samples. The results showed that Rg1 treatment downregulated rectal temperature, udder skin temperature, udder girth, milk somatic cell count, and N-acetyl-β-d-glucosaminidase and upregulated milk production, lactose, and recovered blood components, such as white blood cells, neutrophils, lymphocytes, total proteins, albumin, and globulin. Considering the positive therapeutic effect on lipopolysaccharide-induced mastitis in goats presented in this study as well as the anti-inflammatory activity found previously, the botanical Rg1 deserves further study as a therapeutic agent in the treatment of E. coli mastitis in dairy animals.

Sex influences in behavior and brain inflammatory and oxidative alterations in mice submitted to lipopolysaccharide-induced inflammatory model of depression

Peripheral inflammation induced by lipopolysaccharide (LPS) causes a behavioral syndrome with translational relevance for depression. This mental disorder is twice more frequent among women. Despite this, the majority of experimental studies investigating the neurobiological effects of inflammatory models of depression have been performed in males. Here, we sought to determine sex influences in behavioral and oxidative changes in brain regions implicated in the pathophysiology of mood disorders (hypothalamus, hippocampus and prefrontal cortex - PFC) in adult mice 24 h post LPS challenge. Myeloperoxidase (MPO) activity and interleukin (IL)-1β levels were measured as parameters of active inflammation, while reduced glutathione (GSH) and lipid peroxidation as parameters of oxidative imbalance. We observed that male mice presented behavioral despair, while females anxiety-like alterations. Both sexes were vulnerable to LPS-induced anhedonia. Both sexes presented increased MPO activity in the PFC, while male only in the hippocampus. IL-1β increased in the PFC and hypothalamus of animals of both sexes, while in the hippocampus a relative increase of this cytokine in males compared to females was detected. GSH levels were decreased in all brain areas investigated in animals of both sexes, while increased lipid peroxidation was observed in the hypothalamus of females and in the hippocampus of males after LPS exposure. Therefore, the present study gives additional evidence of sex influence in LPS-induced behavioral alterations and, for the first time, in the oxidative changes in brain areas relevant for mood regulation.

Low incubation temperature during early development negatively affects survival and related innate immune processes in zebrafish larvae exposed to lipopolysaccharide

In many fish species, the immune system is significantly constrained by water temperature. In spite of its critical importance in protecting the host against pathogens, little is known about the influence of embryonic incubation temperature on the innate immunity of fish larvae. Zebrafish (Danio rerio) embryos were incubated at 24, 28 or 32 °C until first feeding. Larvae originating from each of these three temperature regimes were further distributed into three challenge temperatures and exposed to lipopolysaccharide (LPS) in a full factorial design (3 incubation × 3 challenge temperatures). At 24 h post LPS challenge, mortality of larvae incubated at 24 °C was 1.2 to 2.6-fold higher than those kept at 28 or 32 °C, regardless of the challenge temperature. LPS challenge at 24 °C stimulated similar immune-related processes but at different levels in larvae incubated at 24 or 32 °C, concomitantly with the down-regulation of some chemokine and lysozyme transcripts in the former group. Larvae incubated at 24 °C and LPS-challenged at 32 °C exhibited a limited immune response with up-regulation of hypoxia and oxidative stress processes. Annexin A2a, S100 calcium binding protein A10b and lymphocyte antigen-6, epidermis were identified as promising candidates for LPS recognition and signal transduction.

Effects of hydroxychloride and sulfate form of zinc and manganese supplementation on superoxide dismutase activity and immune responses post lipopolysaccharide challenge in poultry fed marginally lower doses of zinc and manganese

Effects of hydroxychloride (OHCl) and sulfate form of zinc and manganese supplementation on immune responses of birds fed marginally lower levels of zinc and manganese during an experimental lipopolysaccharide (LPS) injection were studied. In experiment I, 30-week-old layer birds were fed 50mg/kg Zn+45mg/kg Mn or 100mg/kg Zn+90mg/kg Mn in sulfate or OHCl form and injected with 0 or 500μg/kg LPS in a 2 (50mg Zn+45mg Mn and 100mg Zn+90mg Mn) X 2 (sulfate and OHCl) X 2 (0 and 500μg LPS) factorial setup of treatments for 10 weeks. Among LPS-injected birds, those receiving 50mg ZnOHCl+45mg MnOHCl had comparable heterophil and monocyte superoxide dismutase (SOD) activity compared to the birds fed 100mg Zn+90mg Mn. Compared to the birds injected with PBS, LPS injection upregulated cathelicidin and IL-1 relative mRNA amounts in monocytes from birds fed 100mg Zn+90mg Mn, both in sulfate and OHCl form, and in birds fed 50mg ZnOHCl+45mg MnOHCl, but not in the birds fed 50mg ZnSO4+45mg MnSO4. In experiment II, one-day-old broiler birds were fed 50mg ZnOHCl+45mg MnOHCl, 50mg ZnOHCl+90mg MnOHCl, 100mg ZnOHCL+45mg MnOHCl, 100mg ZnOHCl+90mg MnOHCl, 50mg ZnSO4+45mg MnSO4, or 100mg ZnSO4+90mg MnSO4 for 21 and 42 days. Birds fed 100mg ZnOHCl+45mg MnOHCl form had a comparable heterophil and monocyte SOD activity and monocyte cathelicidin mRNA amounts compared to the group fed 100mg Zn+90mg Mn. Increasing the ZnOHCl content from 50mg to 100mg/kg Zn reversed (P >0.05) the decrease in SOD activity and monocyte cathelicidin mRNA levels of the 50mg ZnOHCl+45mg MnOHCL fed group, and increasing the MnOHCl content from 45mg to 90mg/kg in the 100mg ZnOHCl+45mg MnOHCl group further increased SOD activity. In conclusion, birds fed diets with lower amounts of zinc and manganese in sulfate form decreased SOD activity and IL-1 and cathelicidin amounts during inflammation, and either increasing the dietary zinc and manganese content or feeding zinc and manganese in OHCl form synergistically increased the SOD activity and IL-1 and cathelicidin mRNA amounts in immune cells.

Curcumin inhibits lipopolysaccharide (LPS)-induced endotoxemia and airway inflammation through modulation of sequential release of inflammatory mediators (TNF-α and TGF-β1) in murine model.

Curcumin (diferuloylmethane), a major component of turmeric is well known for its anti-inflammatory potential. Present study investigates sequential release of inflammatory mediators post LPS challenge (10 mg/kg,i.p.) causing lung inflammation and its modulation by curcumin through different routes (20mg/kg, i.p and 10mg/kg, i.n.) in murine model. Dexamethasone (1mg/kg, i.p) was used as standard drug. Lung Inflammation was evaluated by histopathological analysis, myeloperoxidase (MPO) activity followed by inflammatory cell count and total protein content measurements in bronchoalveolar fluid (BALF). Reactive oxygen species (ROS), nitrite and TNF-α levels were measured as markers of endotoxin shock at different time points (1-72h). The mRNA expression of transforming growth factors-β1 (TGF-β1), iNOS and Toll-like receptor-4 (TLR-4) were measured followed by Masson's trichrome staining and hydroxyproline levels as collagen deposition marker leading to fibrotic changes in lungs. We found that LPS-induced lung inflammation and injury was maximum 24-h post LPS challenge shown by MPO and histological analysis which was further supported by elevated nitrite and ROS levels whereas TNF-α level was highest after 1h. Endotoxin-induced mortality was significantly reduced in curcumin (i.p) pretreatment groups up to 72-h post LPS challenge. Significant inhibition in mRNA expression of iNOS, TGF-β1 and TNF-α level was noted after curcumin treatment along with lowered MPO activity, inflammatory cell count, ROS, nitrite levels and collagen deposition in lungs. Our results suggest that higher endotoxin dose causes inflammatory mediator release in chronological order which tend to increase with time and reached maximum after 24-h post-endotoxin (LPS) exposure. Intraperitoneal route of curcumin administration was better in modulating inflammatory mediator release in early phase as compared to intranasal route of administration. It can be used as supplementary therapeutic intervention at early stage of endotoxemia, having fewer side effects.

Effects of immunomodulatory nutrients on growth performance and immune-related gene expression in layer chicks challenged with lipopolysaccharide

Immunomodulatory nutrients alter the immune response to pathogens. This study was conducted to determine the effects of immunomodulatory nutrients on the immune response to a lipopolysaccharide (LPS) challenge of layer chicks fed supplemental corn oil (control; 3%), fish oil (3%), conjugated linoleic acid (CLA; 1%), lutein (0.05 g/kg), or vitamin E (90 I.U./ kg). Four-week-old layer chicks were allotted to 10 treatment groups arranged as a 2 × 5 factorial with 2 sexes and 5 dietary treatments (TRT; 5 replicate pens of 2 male and 2 female chicks per TRT). After a 2-week diet adaptation, all birds were injected intraperitoneal with 1.5 mg/kg BW LPS. Twelve hours post challenge, samples were collected. Fish oil fed birds had greater (P = 0.03) spleen weight (% final BW) than the CLA fed birds. In the liver, the fish oil TRT had higher (P = 0.040) IL-12 expression than the corn oil TRT, but the corn oil TRT had greater (P = 0.001) IL-4 expression than the CLA, lutein, and vitamin E TRT. There was a main effect of sex of the birds on growth parameters at 12 h post LPS challenge in which male birds had greater beginning BW (P < 0.001), final BW (P < 0.001), and greater 12-hour BW loss (P = 0.020) than the female birds, but not relative weight loss. There were also main effects of sex on immune-related gene expression with the females having greater gene expression than the males in the duodenal mucosal scrapings [IL-1β, IL-12, and TLR-4 gene expression (P = 0.026, 0.011, and 0.002, respectively)]; liver [IL-10, IL-4, and iNOS gene expression (P = 0.017, 0.032, and 0.006, respectively)]; and spleen [IL-1β, IL-10, IL-4, and iNOS gene expression (P = 0.001, 0.001, 0.001, and 0.005, respectively)]. Therefore, each immunomodulatory nutrient added to the diets of layer chickens resulted in different immune responses to an LPS challenge.

Immunomodulatory properties of gamithromycin and ketoprofen in lipopolysaccharide-challenged calves with emphasis on the acute-phase response

Macrolide antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to be modulators of the innate immune response, irrespectively of their antimicrobial and anti-inflammatory actions. Therefore, it was our objective to evaluate whether the macrolide gamithromycin (GAM) and the NSAID ketoprofen (KETO) attenuate the acute-phase response in calves, and whether their combined administration is beneficial due to synergistic and/or additive effects. To this end, both drugs, as well as their combination, were studied in a previously developed inflammation model, i.e., the induction of an acute-phase response by an intravenous lipopolysaccharide (LPS) challenge (0.5μg/kg body weight). Sixteen 4-week-old Holstein–Friesian calves were randomized into 4 groups: a positive control (+CONTR) group, receiving LPS but no pharmacological treatment (n=4) and a GAM (n=4), a KETO (n=4) and a GAM–KETO (n=4) group, receiving the respective drugs 1h prior to LPS administration. Clinical scoring and blood collection were performed at regular time points until 72h post LPS challenge. Plasma concentrations of the selected cytokines (tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6)), acute-phase protein (serum amyloid A (SAA)) and prostaglandin E2 (PGE2) were subsequently quantified. Pre-treatment with GAM had no effect in the inflammation model compared to the +CONTR group. KETO, on the other hand, completely inhibited depression, anorexia and fever. This remarkable influence was associated with a significant reduction of PGE2 synthesis by KETO, while the effect on TNF-α, IL-6 and SAA was not straightforward. The combined administration of GAM and KETO provided no synergistic or additive effects in this model, neither clinically nor regarding the studied inflammatory mediators. In conclusion, KETO entirely inhibited PGE2 synthesis, fever development and depression, while GAM did not exert any effect in this model. These results promote the concomitant use of an antimicrobial drug and a NSAID in the treatment of calf diseases associated with LPS, both to enhance clinical recovery and to improve animal welfare.

Study of the immunomodulatory properties of gamithromycin and dexamethasone in a lipopolysaccharide inflammation model in calves

The aim of this study was to define the in vivo immunomodulatory properties of the macrolide antibiotic gamithromycin in calves, with respect to the acute phase response. Additionally, the corticosteroid dexamethasone was included as a positive control immunomodulatory drug. Both drugs, as well as their combination, were studied in a previously developed inflammation model, which was initiated by an intravenous lipopolysaccharide (LPS) challenge (0.5μg/kg body weight). Twenty-four 4-week-old male Holstein Friesian calves were randomized into four groups: no pharmacological treatment (n=6) or a pharmacological treatment with gamithromycin (n=6), dexamethasone (n=6) or their combination (n=6) 1h prior to LPS administration. Blood collection and clinical scoring were performed at regular time points until 72h post LPS challenge. Plasma concentrations of selected cytokines (tumour necrosis factor-α (TNF-α) and interleukin 6 (IL-6)) and acute phase proteins (serum amyloid A and haptoglobin) were subsequently determined. Gamithromycin did not have any beneficial effect on the LPS-induced clinical signs (dyspnea, fever, anorexia and depression), nor on the studied inflammatory mediators. In the dexamethasone and combination groups, the occurrence of dyspnea and fever was not prominently influenced, although the calves recovered significantly faster from the challenge. Moreover, dexamethasone significantly inhibited the levels of TNF-α and IL-6, suggesting a key role for these cytokines in sickness behaviour. In conclusion, unlike dexamethasone, gamithromycin did not directly reduce cytokine release in an LPS inflammation model in calves.

Effect of Inhaled Endotoxin on Mucociliary Clearance and Airway Inflammation in Mild Smokers and Nonsmokers

In healthy nonsmokers, inhaled endotoxin [lipopolysaccharide (LPS)] challenge induces airway neutrophilia and modifies innate immune responses, but the effect on mucociliary clearance (MCC), a key host defense response, is unknown. Although smokers are chronically exposed to LPS through inhaled tobacco smoke, the acute effect of inhaled LPS on both MCC and airway inflammation is also unknown. The purpose of this study was to determine the effect of inhaled LPS on MCC in nonsmokers and mild smokers with normal pulmonary function. We performed an open-label inhalational challenge with 20,000 endotoxin units in healthy adult nonsmokers (n=18) and young adult, mild smokers (n=12). At 4 hr post LPS challenge, we measured MCC over a period of 2 hr, followed by sputum induction to assess markers of airway inflammation. No significant changes in spirometry occurred in either group following LPS challenge. Following LPS, MCC was significantly (p<0.05) slowed in nonsmokers, but not in smokers [MCC=10±9% (challenge) vs. 15±8% (baseline), MCC=14±9% (challenge) vs. 16±10% (baseline), respectively]. Both groups showed a significant (p<0.05) increase in sputum neutrophils 6 hr post LPS challenge versus baseline. Although there was no correlation between the increased neutrophilia and depressed MCC post LPS in the nonsmokers, baseline neutrophil concentration predicted the LPS-induced decrease in MCC in the nonsmokers, i.e., lower baseline neutrophil concentration was associated with greater depression in MCC with LPS challenge (p<0.05). These data show that a mild exposure to endotoxin acutely slows MCC in healthy nonsmokers. MCC in mild smokers is unaffected by mild endotoxin challenge, likely due to preexisting effects of cigarette smoke on their airway epithelium.