Low-dose Lipopolysaccharide Research Articles

Effect on the splenocyte function of weaned piglets induced by continuous lipopolysaccharide injections.

When piglets are exposed to pathogens for a long period, the immune system organs, among them the spleen, play a major role in combating the stress caused by those pathogens. In the present study, the effect on splenocyte function was investigated in a model of weaned piglets in which stress was induced by multiple low doses of lipopolysaccharide (LPS). Forty-eight 28-day-old piglets were divided into two groups: the LPS group and the control group. During the experimental period of thirteen days, the LPS group was intraperitoneally injected with LPS (100 μg/kg) once per day, and the control group was injected with the same volume of 0.9% sterile saline. On the 1st, 5th, 9th and 13th days, the piglets' spleens were collected for isolating splenocytes. The proliferation ability of splenocytes was evaluated by the cell-counting-kit 8 method. Flow cytometry was used to detect cell cycle stage and apoptosis, and the nitric oxide level of cell supernatant was also tested. In the experimental group, the proliferation ability of splenocytes was enhanced, the proportion of cells in the G0/G1 phase was smaller, and cells were promoted to the S and G2/M phases. Meanwhile, apoptosis was suppressed and nitric oxide release upregulated. The results were significantly different between the LPS group and the control group on the 5th and 9th days. The difference between the results of one group and those of the other suggest that after the 5th LPS injection, multiple low doses of LPS activated splenocytes and restored the number of splenocytes, which maintained and possibly enhanced the regulation of the immune function of the spleen.

Differential Responses of Spinal Microglia to Low-Dose LPS Delivered in the Daily Rest vs Active Phase

Virtually all lung and neural disorders are associated with systemic and/or neuro-inflammation. Neuro-inflammation impairs important forms of neuroplasticity associated with cognitive and spinal motor learning ( e.g., respiratory motor plasticity). Microglia are resident CNS innate immune cells that regulate diverse processes including neuroinflammation and multiple forms of neuroplasticity via neuron-microglia interactions. Brain microglia exhibit profound diurnal rhythms in their response to pro-inflammatory stimuli, such as toll-like receptor 4 (TLR4) activation. Although hippocampal microglia develop a pro-inflammatory phenotype in response to TLR4 activation during the daily rest phase, little is known concerning spinal microglial responses to inflammatory stimuli across the daily rest/active cycle. Thus, we tested the hypothesis that ventral spinal microglia near the phrenic motor nucleus differentially respond to low-dose systemic lipopolysaccharide (LPS) delivered in the rest (12PM) vs active (12AM) phase. Specifically, we investigated microglial responses to systemic inflammation induced by low-dose LPS (0.1mg/kg, i.p.) in 2 populations of adult Sprague-Dawley rats (both 3-6 months old): 1) SD wildtype and 2) SD rats with humanized neuro-neutral apolipoprotein 3 (h APOE3) knock-in. All rats received intrapleural Cholera toxin B (CtB) injections 2 weeks prior to harvest to identify phrenic motor neurons. Twenty-four hours post-LPS or vehicle (0.9% saline), rats were perfused with 1X PBS and fixed with 4% PFA. Spinal tissues containing the phrenic motor nucleus (cervical 3-6) were harvested and dehydrated in 30% sucrose for immunohistochemistry. Activated microglia near phrenic motor neurons were visualized with an Iba-1 antibody and quantified and phenotyped based on morphology. Since no differences in microglia were found between wildtype and h APOE3 rats, data were combined into a single group for analyses. In vehicle-treated rats, robust Iba-1 staining near phrenic motor neurons was observed in the active, but not rest phase. In contrast, Iba-1 staining in LPS treated rats was elevated only when LPS was administered in the rest (not active) phase. After rest-phase LPS injections, microglial cell bodies were larger and had more processes compared to rest-phase controls. In the active phase, spinal microglia were similar between vehicle and LPS injected rats. Thus, spinal microglia exhibit a profound diurnal rhythm in (morphological) responses to systemic inflammation ( i.e. LPS). Further studies are needed to identify specific molecules driving time-of-day differences in spinal microglial responses to pro-inflammatory stimuli. Nevertheless, these findings increase our understanding of how diurnal rhythms impact cellular responses to physiological challenges, such as inflammation. Further, our results yield clues concerning diurnal variations in phrenic motor plasticity, since reciprocal phrenic motor neuron/microglial communication is an important regulator of phrenic motor plasticity. NIH R01HL148030 (GSM), NIH R01HL149800 (GSM), NIH T32HL134621-5 (ABM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ginsenoside Rg1 attenuates chronic inflammation-induced renal fibrosis in mice by inhibiting AIM2 inflammasome in an Nrf2-dependent manner

Chronic kidney disease (CKD) is a growing global public health issue. Ginsenoside Rg1 (Rg1) has favorable nephroprotective effects, but its role and mechanism in CKD need further investigation. Therefore, we observed the effect of Rg1 on the mouse kidneys after 21 days of low-dose lipopolysaccharide (LPS) exposure. Meanwhile, we investigated the protective mechanism of Rg1 in LPS-induced mesangial cells using the Nrf2 inhibitor ML385. We found that chronic exposure to LPS caused fibrosis in mouse kidneys, accompanied by AIM2 activation and oxidative stress imbalance; however, Rg1 restored the anomalies, accompanied by activation of Nrf2/HO-1 signaling. Finally, our in vitro studies revealed that Rg1′s nephroprotective effects could be inhibited by ML385, and Rg1 can bind Nrf2, suggesting that the protective effects of Rg1 may involve Nrf2 activation and then inhibition of the AIM2. Our study has significant implications for the prevention and treatment of kidney diseases, particularly those related to inflammation.

Endotoxin tolerance ameliorates lipopolysaccharide/D-galactosamine-induced acute liver failure by negative regulation of the NF-κB/NLRP3 and activation of Nrf2/HO-1 via Sitr1

Acute liver failure (ALF) is a potentially fatal disorder characterized by extensive hepatocyte necrosis and rapid decline in liver function. Numerous factors, including oxidative stress, cell death, and inflammatory responses, are associated with its pathogenesis. Endotoxin tolerance (ET) refers to the phenomenon in which the body or cells exhibit low or no response to high-dose lipopolysaccharide (LPS) stimulation after pre-stimulation with low-dose LPS. However, the specific mechanism through which ET regulates LPS/D-galactosamine (D-GalN)-induced ALF remains unclear. An ALF mouse model was established by intraperitoneal injection of D-GalN (400 mg/kg) and LPS (10 mg/kg). A low dose of LPS (0.1 mg/kg/d) was continuously administered to mice for 5 d before modeling to assess the protective effect of ET. The data from this study showed that ET alleviated the inflammatory response in mice with LPS/D-GalN-induced ALF. ET inhibited LPS-induced oxidative damage and pyroptosis in macrophages in vitro. RNA sequencing analysis showed that the NF-κB/NLRP3 pathway was linked to the anti-inflammatory and antioxidative effects of ET. Furthermore, using western blot, RT-qPCR, and immunofluorescence, we verified that ET inhibited the NF-κB/NLRP3 pathway and triggered the Nrf2/HO-1 signaling pathway to attenuate oxidative stress and cell pyroptosis. Sirt1 knockdown reversed this protective effect. In summary, our research elucidates that ET prevents ALF advancement by upregulating Sirt1 levels, triggering the Nrf2/HO-1 signaling axis, and suppressing the NF-κB/NLRP3 signaling cascade to inhibit oxidative stress and cell pyroptosis. Our results provide a mechanistic explanation for the protective effect of ET against ALF.

Mitochondrial dysfunction precedes hippocampal IL-1β transcription and cognitive impairments after low-dose lipopolysaccharide injection in aged mice

Acute cognitive impairments termed delirium often occur after inflammatory insults in elderly patients. While previous preclinical studies suggest mitochondria as a target for reducing neuroinflammation and cognitive impairments after LPS injection, fewer studies have evaluated the effects of a low-grade systemic inflammation in the aged brain. Thus, to identify the significance of mitochondrial dysfunction after a clinically relevant systemic inflammatory stimulus, we injected old-aged mice (18–20 months) with low-dose lipopolysaccharide (LPS, 0.04 mg/kg). LPS injection reduced mitochondrial respiration in the hippocampus 24 h after injection (respiratory control ratio [RCR], state3u/state4o; control = 2.82 ± 0.19, LPS = 2.57 ± 0.08). However, gene expression of the pro-inflammatory cytokine IL-1β was increased (RT-PCR, control = 1.00 ± 0.30; LPS = 2.01 ± 0.67) at a more delayed time point, 48 h after LPS injection. Such changes were associated with cognitive impairments in the Barnes maze and fear chamber tests. Notably, young mice were unaffected by low-dose LPS, suggesting that mitochondrial dysfunction precedes neuroinflammation and cognitive decline in elderly patients following a low-grade systemic insult. Our findings highlight mitochondria as a potential therapeutic target for reducing delirium in elderly patients.

Acute experimental inflammation in healthy women attenuates empathy for psychological pain

Administration of low-dose lipopolysaccharide (LPS) to healthy humans is a translational approach to analyze the effects of acute systemic inflammation and sickness behavior. Although studies documented that LPS-induced inflammation can alter social behavior, its impact on empathy remains poorly understood. In this double-blind, placebo-controlled study, 52 healthy female volunteers received an intravenous injection of either LPS (0.4 ng/kg body weight) or placebo and completed the Social Interaction Empathy Task (SIET) two hours after injection. Physiological responses (blood pressure, heart rate, body temperature, cytokines, cortisol) were analyzed along with sickness symptoms and mood before and after LPS or placebo administration. LPS application led to significant increases in plasma cytokines and sickness symptoms as well as low mood. Moreover, volunteers receiving LPS showed significantly less empathy for other’s psychological pain than those who received placebo. Furthermore, LPS-injected volunteers with more severe sickness symptoms displayed higher pain ratings in the first-person perspective. Thus, low-grade inflammation reduces empathy for other’s psychological pain which might reflect an adaptive strategy to save energy by not responding empathetically when sick oneself.

Imaging quantitative changes in blood-brain barrier permeability using [18F]2-fluoro-2-deoxy-sorbitol ([18F]FDS) PET in relation to glial cell recruitment in a mouse model of endotoxemia

The quantitative relationship between the disruption of the blood-brain barrier (BBB) and the recruitment of glial cells was explored in a mouse model of endotoxemia. [18F]2-Fluoro-2-deoxy-sorbitol ([18F]FDS) PET imaging was used as a paracellular marker for quantitative monitoring of BBB permeability after i.v injection of increasing doses of lipopolysaccharide (LPS) or vehicle (saline, n = 5). The brain distribution of [18F]FDS (VT, mL.cm−3) was estimated using kinetic modeling. LPS dose-dependently increased the brain VT of [18F]FDS after injection of LPS 4 mg/kg (5.2 ± 2.4-fold, n = 4, p < 0.01) or 5 mg/kg (9.0 ± 9.1-fold, n = 4, p < 0.01) but not 3 mg/kg (p > 0.05, n = 7). In 12 individuals belonging to the different groups, changes in BBB permeability were compared with expression of markers of astrocyte (GFAP) and microglial cell (CD11b) using ex vivo immunohistochemistry. Increased expression of CD11b and GFAP expression was observed in mice injected with 3 mg/kg of LPS, which did not increase with higher LPS doses. Quantitative [18F]FDS PET imaging can capture different levels of BBB permeability in vivo. A biphasic effect was observed with the lowest dose of LPS that triggered neuroinflammation without disruptive changes in BBB permeability, and higher LPS doses that increased BBB permeability without additional recruitment of glial cells.

Low-Dose LPS Modulates Microglia/Macrophages Phenotypic Transformation to Amplify Rehabilitation Effects in Chronic Spinal Cord Injured (CSCI) Mice.

Spinal cord injury (SCI) results in stalled motor function recovery under the chronic phase. One of the reasons due to the presence of ongoing inflammation. Therefore, regulating the status of immune cells may help reopen the window for neural repair, which represents a potential therapeutic target. In this study, we aimed to investigate whether this could be achieved in mice with cervical 5 crush CSCI (4W) by utilizing a concentration of 0.5mg/kg of lipopolysaccharide (LPS) to stimulate microglia/macrophages. Additionally, the mice underwent rehabilitation training for another 6 weeks. Our results showed that systemic injection of LPS enhanced the effects of forelimb rehabilitation training, as evaluated through single pellet grasping (SPG). Electrophysiological studies revealed the restoration of cortical drive to the injured side's forelimb muscles in the training combined with LPS group. Tract tracing studies demonstrated the reconstruction of cortical innervation to the cervical spinal cord. Furthermore, the levels of pro-inflammatory phenotype markers, such as inducible nitric oxide synthase (INOS) and CD68, decreased, while the expression of anti-inflammatory phenotype markers, including arginase 1 (ARG-1) and CD206, increased. Importantly, this phenotypic switch in microglia/macrophages was accompanied by an increase in phagocytic activity markers as indicated by BODIPY + IBA1 + staining. Collectively, our data suggests that low-dose LPS improves the effects of rehabilitation training by regulating the phenotypic transformation of microglia/macrophages in CSCI.This study provides a fresh perspective and intervention direction for the clinical treatment of chronic spinal cord injuries.

High variation in the response of calves to a low-dose lipopolysaccharide challenge is associated with early-life measurements

Lipopolysaccharide (LPS) challenges are commonly used in animal studies as a model for infection with gram-negative bacteria and innate immune activation. We used a low-dose LPS challenge for evaluating interindividual variation in innate immune responses in calves. This was part of a larger study aimed at predicting interindividual variation in feed efficiency in veal calves by variation in feeding motivation, digestion, metabolism, immunology, and behavioral traits. However, due to unexpected high mortality, this LPS challenge was performed in 32 calves rather than in 130 calves, which was initially intended in that larger study, and the 32 calves subjected to the LPS challenge were removed from that larger study. The objective of this short communication is to report the effects of a low-dose LPS challenge in those 32 calves and to examine whether the high variation in calves' responses to LPS could be explained by parameters related to feeding motivation, digestion, behavior, and immunology measured in early life. Thirty-two male Holstein-Friesian calves of Dutch origin were intravenously injected with LPS (0.05 μg/kg of body weight) at an age of 72 ± 0.6 d. Rectal temperature and respiratory frequency were recorded before injection and every hour after injection up to 6 h. In the 8 wk before the LPS challenge, measurements were performed related to general health, feeding motivation, digestion, behavior, and immunity. Following LPS administration, 3 calves died of shock, a fourth calf was euthanized because of severe symptoms of shock and 3 other calves were treated with corticosteroids to counteract observed symptoms of shock. Within the group of 25 relatively mild-responding calves, large interindividual variation in clinical responses to LPS was observed. The maximum increase in rectal temperature varied from 0.6 to 1.9°C and averaged 1.2 ± 0.39°C (coefficient of variation was 32%). The maximum increase in respiratory frequency varied from 16 to 132 bouts/min and averaged 60 ± 28 bouts/min (coefficient of variation was 48%). Little differences were found in early-life measurements between the 7 heavy and 25 mild responders, although heavy responders tended to have a better umbilical hernia score, and had a lower score in a human approach test (i.e., were less reactive) and lower presence of fecal pathogens. The maximum increase in rectal temperature correlated negatively with blood hemoglobin concentration at arrival of the calves at the facilities (r = −0.59) and in wk 4 (r = −0.53). The maximum increase in respiratory frequency correlated negatively with fecal color score (r = −0.43) and positively with fur score in wk 5 (r = 0.50). Overall, mortality (12.5%) and variation in clinical response was high after a low-dose LPS challenge in clinically healthy calves and some hematological and health measurements in early life were related to the clinical response of calves to LPS.

RAGE in circulating immune cells is fundamental for hippocampal inflammation and cognitive decline in a mouse model of latent chronic inflammation

BackgroundLatent chronic inflammation has been proposed as a key mediator of multiple derangements in metabolic syndrome (MetS), which are increasingly becoming recognized as risk factors for age-related cognitive decline. However, the question remains whether latent chronic inflammation indeed induces brain inflammation and cognitive decline. MethodsA mouse model of latent chronic inflammation was constructed by a chronic subcutaneous infusion of low dose lipopolysaccharide (LPS) for four weeks. A receptor for advanced glycation end products (RAGE) knockout mouse, a chimeric myeloid cell specific RAGE-deficient mouse established by bone marrow transplantation and a human endogenous secretory RAGE (esRAGE) overexpressing adenovirus system were utilized to examine the role of RAGE in vivo. The cognitive function was examined by a Y-maze test, and the expression level of genes was determined by quantitative RT-PCR, western blot, immunohistochemical staining, or ELISA assays. ResultsLatent chronic inflammation induced MetS features in C57BL/6J mice, which were associated with cognitive decline and brain inflammation characterized by microgliosis, monocyte infiltration and endothelial inflammation, without significant changes in circulating cytokines including TNF-α and IL-1β. These changes as well as cognitive impairment were rescued in RAGE knockout mice or chimeric mice lacking RAGE in bone marrow cells. P-selectin glycoprotein ligand-1 (PSGL-1), a critical adhesion molecule, was induced in circulating mononuclear cells in latent chronic inflammation in wild-type but not RAGE knockout mice. These inflammatory changes and cognitive decline induced in the wild-type mice were ameliorated by an adenoviral increase in circulating esRAGE. Meanwhile, chimeric RAGE knockout mice possessing RAGE in myeloid cells were still resistant to cognitive decline and brain inflammation. ConclusionsThese findings indicate that RAGE in inflammatory cells is necessary to mediate stimuli of latent chronic inflammation that cause brain inflammation and cognitive decline, potentially by orchestrating monocyte activation via regulation of PSGL-1 expression. Our results also suggest esRAGE-mediated inflammatory regulation as a potential therapeutic option for cognitive dysfunction in MetS with latent chronic inflammation.

PSV-5 The Effect of a Prenatal Immune Challenge on Offspring Skeletal Muscle Characteristics in the Longissimus Muscle of Weaned Pigs

Abstract An insult to the health status of an animal is associated with decreased skeletal muscle growth. Furthermore, an immune challenge to a gestating dam may inhibit pre- and postnatal muscle growth of the offspring. The objective of this study was to determine if the administration of low-dose lipopolysaccharide (LPS) to gestating sows would alter muscle characteristics of the longissimus muscle (LM) in weaned pigs exposed to LPS. Pregnant Camborough sows were randomly assigned to receive LPS (LPS; n = 7) at a dose of 2.5 μg/kg BW or saline (CON; n= 7) on 78 ± 1.8 d of gestation. At weaning (21 ± 1.3 d of age), barrows (CON n = 17; LPS n = 17) from each sow received a secondary LPS challenge. Following the postnatal challenge, barrows were euthanized (31 ± 1.3 d of age), and both LM were collected. The left LM was weighed, measured for length, and measured for circumference at the cranial, middle, and caudal portion of the muscle. Two samples from the right LM were preserved for histological analyses. Morphometric, fiber type count, primary myofiber clustering, and satellite cell counts were analyzed using the MIXED procedure of SAS 9.4 with a main effect of prenatal treatment and covariate of body weight. There were no differences (P &amp;gt; 0.248) between any morphometric measures. The average diameter for myosin heavy chain (MHC) type I and IIB/X fibers was increased (P &amp;lt; 0.048) 8.6 and 5.9% respectively in LPS offspring compared with CON offspring. Average cross-sectional area (CSA) was increased 10.6% (P = 0.030) in MHC IIB/X fibers and tended to be increased 14.9% (P = 0.080) in MHC 1 fibers of LPS offspring. Number of myofibers within the LM was increased 16.1% (P = 0.032) in CON compared with LPS offspring. Diameter, CSA, and total CSA of oxidative myofibers (MHC type I and IIA) clusters was increased (P &amp;lt; 0.001) 13.8, 27.6, and 22.3% respectively in LPS compared with CON offspring. There were no differences (P &amp;gt; 0.186) between treatment groups for total nuclei or nuclei positive for MYF5, PAX7, or MYF5 and PAX7 nuclei. Based on the timing of the maternal challenge, it can be theorized that LPS hinders hyperplasia during secondary myogenesis following an immune challenge to the dam, with a rescue response of increased hypertrophy of MHC I and IIB/X myofibers post-weaning and glycolytic myofiber profile. However, due to the reduction in total myofibers, the impact of LPS on parameters such as average daily gain, feed efficiency, and lean skeletal muscle accretion should be studied further.

34 Assessing the Ability to Immunomodulate the Innate Immune System and Oxidative Status of Weaned Pigs Through a Prenatal Lipopolysaccharide Challenge

Abstract Gastrointestinal immunity and antioxidant defenses may be bolstered in young animals through prenatal immune system stimulation (PIS), but this is largely uninvestigated in swine. The objective of this experiment was to determine if a low dose of lipopolysaccharide (LPS) administered to late-gestating sows would alter the immune response and oxidative status of subsequent offspring. On d 78 ± 1.8 of gestation, 14 Camborough sows (Parity = 2.6±1.4) of the same farrowing group were blocked by body weight and randomly assigned to one of two treatments: intramuscular injection of saline (CON) or LPS from E. coli O111:B4 (2.5 µg/kg of BW). Sow rectal temperatures (RT) and sickness behavior scores (SBS) were recorded every 0.5 h from -0.5 h to 8 h and every 8 h from 8 to 48 h post-treatment. A subset of 34 weaned barrows (n = 17 CON, LPS; weaning age 21 ± 1.3 d) from the experimental sow group were transported on d -7 to an environmentally controlled facility where they were individually housed with ad libitum access to water and feed. On d -1, pigs were anesthetized for placement of a subcutaneous temperature logger and a jugular catheter. On d 0, all pigs were challenged intravenously with the same LPS strain (10 µg/kg of BW) previously administered to sows. Body temperature was measured at 5-min intervals from the time of logger placement. Blood samples were collected at -2, 0, 1, 2, 4, 6, 8, 12, and 24 h relative to LPS administration at 0 h. After the 24 h blood collection, pigs were necropsied for ileal and jejunal tissue. Plasma samples, jejunal and ileal tissues were analyzed for total antioxidant capacity (TAC) and malondialdehyde (MDA). Data were analyzed as a linear mixed model using PROC MIXED (SAS 9.4) with treatment and time as fixed effects. A dependent covariance structure was used for repeated measurements. Sows administered LPS had elevated RT from 2 to 16-h post-challenge with return to baseline by h 24, indicating mild immune stimulation (treatment × time; P &amp;lt; 0.01). Similarly, sows exposed to LPS had increased SBS from 2 to 3.5 h post-challenge (treatment × time; P &amp;lt; 0.01). Pigs exposed to PIS had greater body temperature than CON at 1 and 2 h post-LPS (treatment × time; P &amp;lt; 0.01). An increase in plasma TAC at 2 and 4 h post-LPS was observed in pigs exposed to PIS relative to CON (treatment × time; P &amp;lt; 0.01). Irrespective of treatment, LPS increased plasma MDA from 2 to 24 h post-challenge (time; P &amp;lt; 0.01). There was no effect of PIS on ileal or jejunal tissue TAC (P ≥ 0.40), but weaned pigs exposed to PIS had decreased jejunal MDA (P &amp;lt; 0.05). Collectively, it appears PIS may alter the innate immune response and oxidative status of the weaned pig during LPS challenge.

A Translational Model for Repeated Episodes of Joint Inflammation: Welfare, Clinical and Synovial Fluid Biomarker Assessment.

This study investigates repeated low-dose lipopolysaccharide (LPS) injections in equine joints as a model for recurrent joint inflammation and its impact on animal welfare. Joint inflammation was induced in eight horses by injecting 0.25 ng of LPS three times at two-week intervals. Welfare scores and clinical parameters were recorded at baseline and over 168 h post-injection. Serial synoviocentesis was performed for the analysis of a panel of synovial fluid biomarkers of inflammation and cartilage turnover. Clinical parameters and a final synoviocentesis were also performed eight weeks after the last sampling point to assess the recovery of normal joint homeostasis. Statistical methods were used to compare the magnitude of response to each of the 3 LPS inductions and to compare the baseline and final measurements. Each LPS injection produced consistent clinical and biomarker responses, with minimal changes in welfare scores. General matrix metalloproteinase (MMP) activity and joint circumference showed greater response to the second LPS induction, but response to the third was comparable to the first. Gylcosaminoglycans (GAG) levels showed a significantly decreased response with each induction, while collagen-cleavage neoepitope of type II collagen (C2C) and carboxypropetide of type II collagen epitope (CPII) showed quicker responses to the second and third inductions. All parameters were comparable to baseline values at the final timepoint. In conclusion, a consistent, reliable intra-articular inflammatory response can be achieved with repeated injections of 0.25 ng LPS, with minimal impact on animal welfare, suggesting potential as a refined translational model of recurrent joint inflammation.

Low-dose lipopolysaccharide exposure during oocyte maturation disrupts early bovine embryonic development

Gram-negative bacteria release of lipopolysaccharide (LPS) endotoxin elicits robust immune responses capable of disrupting normal ovarian function contributing to female infertility. However, effects of subclinical or non-detectable infections on oocyte competence and subsequent embryo development remain to be fully elucidated. The aim of this study was to investigate the effects of exposing bovine oocytes to low LPS doses on oocyte and embryo competence. Bovine oocytes were collected from slaughterhouse-derived ovaries and matured with vehicle-control or increasing doses of LPS (0.01, 0.1, and 1 μg/mL) for 21 h. Oocytes (n = 252) were evaluated for nuclear maturation. A set of embryos from LPS-matured oocytes (n = 300) were cultured for 8 d to evaluate day 3 cleavage rates and day 8 blastocyst rates along with blastocyst cell counts. A subset of oocytes (n = 153) was fertilized and cultured for time-lapse image capture and analysis of embryo development. Results demonstrate no significant treatment differences among treatment groups in percent of oocytes at germinal vesicle (GV; P = 0.90), germinal vesicle breakdown (GVBD; P = 0.13), meiosis I (MI; P = 0.26), or metaphase II (MII; P = 0.44). Likewise, treatment differences were not observed in cleavage rates (P = 0.97), or blastocyst rates (P = 0.88) evaluated via traditional microscopy. Treatment with LPS did not affect total blastocyst cell count (P = 0.68), as indicated by trophectoderm (P = 0.83), and inner cell mass (P = 0.21) cell counts. Time-lapse embryo evaluation demonstrated no differences among control or LPS matured oocytes in number of zygotes that did not cleave after fertilization (P = 0.84), or those that cleaved but arrested at the 2-cell stage (P = 0.50), 4-cell (P = 0.76), prior to morula (P = 0.76). However, embryos derived from oocytes challenged with 0.1 μg/mL LPS tended to have reduced development to the morula stage compared with vehicle-treated controls (P = 0.06). Additionally, the percentage of blastocysts derived from oocytes matured in 0.01 μg/mL LPS tended to decrease compared to vehicle-treated controls (11.38 and 25.45 %, respectively; P = 0.09). Similarly, the proportion of oocytes that developed to the blastocyst stage was greater in vehicle-treated controls (25.45 %) compared with embryos derived from oocytes matured in 0.1 and 1 μg/mL (5.92 and 6.55 %, respectively; P = 0.03) LPS. These data suggest LPS-matured oocytes that subsequently underwent in vitro fertilization, experienced decreased competence to develop to the blastocyst stage.

Inter-alpha inhibitor proteins attenuate lipopolysaccharide-induced blood-brain barrier disruption in neonatal mice

There is a paucity of information regarding efficacious pharmacological neuroprotective strategies to attenuate or reduce brain injury in neonates. Lipopolysaccharide (LPS) disrupts blood-brain barrier (BBB) function in adult rodents and increases inflammation in adults and neonates. Human blood-derived Inter-alpha Inhibitor Proteins (IAIPs) are neuroprotective, improve neonatal survival after LPS, and attenuate LPS-induced disruption of the BBB in adult male mice. We hypothesized that LPS also disrupts the function of the BBB in neonatal mice and that IAIPs attenuate the LPS-induced BBB disruption in male and female neonatal mice. IAIPs were administered to neonatal mice after LPS and BBB permeability quantified with intravenous 14C-sucrose and 99mTc-albumin. Although repeated high doses (3 mg/kg) of LPS in neonates resulted in high mortality rates and a robust increase in BBB permeability, repeated lower doses (1 mg/kg) of LPS resulted in lower mortality rates and disruption of the BBB in both male and female neonates. IAIP treatment attenuated disruption of the BBB similarly to sucrose and albumin after exposure to low-dose LPS in neonatal mice. Exposure to low-dose LPS elevated IAIP concentrations in blood, but it did not appear to increase the systemic levels of Pre-alpha inhibitor (PaI), one of the family members of the IAIPs that contains heavy chain 3. We conclude that IAIPs attenuate LPS-related disruption of the BBB in both male and female neonatal mice.

Utility of In Vitro Cellular Models of Low-Dose Lipopolysaccharide in Elucidating the Mechanisms of Anti-Inflammatory and Wound-Healing-Promoting Effects of Lipopolysaccharide Administration In Vivo.

Lipopolysaccharide (LPS) is a bacterial component that activates intracellular signaling pathways upon binding to the Toll-like receptor (TLR)-4/MD-2 complex. It is well known that LPS injected into animals and high-dose (100 ng/mL to 1 μg/mL) LPS treatment to innate immune cells induce an inflammatory response. In contrast, LPS is naturally present in the gastrointestinal tract, respiratory tract, and skin of humans and animals, and it has been shown that TLR-4-deficient animals cannot maintain their immune balance and gut homeostasis. LPS from commensal bacteria can help maintain homeostasis against mucosal stimulation in healthy individuals. Oral LPS administration has been shown to be effective in preventing allergic and lifestyle-related diseases. However, this effect was not observed after treatment with LPS at high doses. In mice, oral LPS administration resulted in the detection of LPS at a low concentration in the peritoneal fluid. Therefore, LPS administered at low and high doses have different effects. Moreover, the results of in vitro experiments using low-dose LPS may reflect the effects of oral LPS administration. This review summarizes the utility of in vitro models using cells stimulated with LPS at low concentrations (50 pg/mL to 50 ng/mL) in elucidating the mechanisms of oral LPS administration. Low-dose LPS administration has been demonstrated to suppress the upregulation of proinflammatory cytokines and promote wound healing, suggesting that LPS is a potential agent that can be used for the treatment and prevention of lifestyle-related diseases.

Low-dose lipopolysaccharide inhibits spinal cord injury-induced neuronal apoptosis by regulating autophagy through the lncRNA MALAT1/Nrf2 axis.

Spinal cord injury (SCI) is a neurological disease associated with a high disability rate. Low-dose lipopolysaccharide (LPS) has been reported to activate cross-immune tolerance and alleviate the effects of various traumatic stimuli. The present study aimed to explore the effect of LPS on SCI and the potential molecular mechanism. Male Sprague-Dawley (SD) rats were used to established an in vivo SCI model and were intraperitoneally injected with lentivirus particles encoding a MALAT1 small interfering RNA (siRNA) on day 10 prior to SCI and with 0.2 mg/kg LPS 72 h prior to SCI. Basso, Beattie, and Bresnahan (BBB) scoring; HE staining; and TUNEL assay were used to assess neurological function and pathophysiological changes. Western blot and immunohistochemistry (IHC) were used to detect cell autophagy and Nrf2 nuclear translocation. PC12 cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to establish an in vitro SCI model. In vitro SCI model cells were pretreated with LPS and transfected with siMALAT1 or MALAT1 overexpression plasmid aimed at knocking down MALAT1 or overexpressing MALAT1. The cell counting kit-8 (CCK-8) assay was used to measure the toxicity of LPS towards PC12 cells. Flow cytometry and immunofluorescence analysis were performed to investigate cell apoptosis and Nrf2 nuclear translocation. SCI rats preconditioned with low-dose LPS had higher BBB scores, reduced SCI injury, increased MALAT1 expression and activated autophagy and Nrf2 nuclear translocation in the in vivo SCI model. In the in vitro SCI model, low-dose LPS treatment suppressed the apoptotic ratio of PC12 cells, increased MALAT1 expression, activated autophagy, and promoted Nrf2 nuclear translocation. Silencing MALAT1 exacerbated OGD/R injury in vitro and weakened the protective effect of low-dose LPS. Overexpression of MALAT1 inhibits OGD/R-induced apoptosis by inducing autophagy and promoting Nrf2 nuclear translocation. This was also been confirmed in animal experiments, silencing MALAT1 blocked the promotion of Nrf2 by low-dose LPS and the alleviated of SCI apoptosis. Low-dose LPS exhibited a protective role on SCI by activating autophagy and suppressing nerve cell apoptosis via the lncRNA MALAT1/Nrf2 axis.

An oral cholera vaccine in the prevention and/or treatment of inflammatory bowel disease.

The oral cholera vaccine WC-rBS consists of 4 different inactivated strains of Vibrio cholerae (LPS source) admixed with recombinant cholera toxin B subunit. Because of its unique composition and anti-inflammatory properties reported for both CTB and low doses of LPS from other Gram-negative bacteria, we speculated that WC-rBS might have anti-inflammatory potential in a chronic autoimmune disease such as inflammatory bowel diseases. First in vitro endotoxin tolerance experiments showed the surprising WC-rBS potential in the modulation of inflammatory responses on both PBMCs and THP1 cells. WC-rBS was further evaluated in the Dextran Sodium Sulfate colitis mouse model. Administrated orally at different dosages, WC-rBS vaccine was safe and showed immunomodulatory properties when administered in a preventive mode (before and during the induction of DSS colitis) as well as in a curative mode (after colitis induction); with improvement of disease activity index (from 27 to 73%) and histological score (from 65 to 88%). Interestingly, the highest therapeutic effect of WC-rBS vaccine was observed with the lowest dosage, showing even better anti-inflammatory properties than mesalamine; an approved 5-aminosalicylic acid drug for treating IBD patients. In summary, this is the first time that a prophylactic medicine, safe and approved for prevention of an infectious disease, showed a benefit in an inflammatory bowel disease model, potentially offering a novel therapeutic modality for IBD patients.

Low-dose LPS alleviates early brain injury after SAH by modulating microglial M1/M2 polarization via USP19/FOXO1/IL-10/IL-10R1 signaling

BackgroundLow-dose lipopolysaccharide (LPS) protects against early brain injury (EBI) after subarachnoid hemorrhage (SAH). However, the mechanism underlying the neuroprotective roles of low-dose LPS remain largely undefined. MethodsA SAH mice model was established and the pathological changes of brain were evaluated by wet-dry weight method, HE and Nissl staining, and blood-brain barrier (BBB) permeability assay. Cell apoptosis and inflammation were monitored by TUNEL, flow cytometry and ELISA assays. qRT-PCR, immunofluorescence and Western blot were used to detect the expression of microglial polarization-related or oxidative stress-associated markers. Bioinformatics analysis, luciferase and ChIP assays were employed to detect the direct association between FOXO1 and IL-10 promoter. The ubiquitination of FOXO1 in the in vitro SAH model was detected by co-IP. ResultsLow-dose LPS alleviated SAH-induced neurological dysfunction, brain edema, BBB disruption, damage in the hippocampus, neuronal apoptosis and inflammation via modulating microglial M1/M2 polarization by IL-10/IL-10R1 signaling. Mechanistic studies showed that FOXO1 acted as a transcriptional activator of IL-10. USP19 mediated the deubiquitination of FOXO1 to activate IL-10/IL-10R1 signaling, thereby regulating microglial M1/M2 polarization. Functional experiments revealed that low-dose LPS upregulated USP19 to modulate microglial M1/M2 polarization via FOXO1/IL-10/IL-10R1 signaling in SAH mice. ConclusionLow-dose LPS protected against EBI after SAH by modulating microglial M1/M2 polarization via USP19/FOXO1/IL-10/IL-10R1 signaling.

Low-dose Lipopolysaccharide Alleviates Spinal Cord Injury-induced Neuronal Inflammation by Inhibiting microRNA-429-mediated Suppression of PI3K/AKT/Nrf2 Signaling.

This study investigated the impact of low-dose lipopolysaccharide (LPS) on spinal cord injury (SCI) and the potential molecular mechanism. Rats were randomly assigned to four groups: Sham, SCI, SCI + LPS, and SCI + LPS + agomir. Allen's weight-drop method was used to establish an in vivo SCI model. The Basso Bcattie Bresnahan rating scale was employed to monitor locomotor function. An in vitro SCI model was constructed by subjecting PC12 cells to oxygen and glucose deprivation/ reoxygenation (OGD/R). Enzyme-linked immunosorbent assay (ELISA) was applied for the determination interleukin (IL)-1β and IL-6. The dual luciferase reporter assay was used to validate the targeting of microRNA (miR)-429 with PI3K. Immunohistochemical staining was used to assess the expression of PI3K, phosphorylated AKT and Nrf2 proteins. The Nrf2-downstream anti-oxidative stress proteins, OH-1 and NQO1, were detected by western blot assay. MiR-429 expression was detected by fluorescence in situ hybridization and real-time quantitative reverse transcription PCR. In vitro, low-dose LPS decreased miR-429 expression, activated PI3K/AKT/Nrf2, inhibited oxidative stress and inflammation, and attenuated SCI. MiR-429 was found to target and negatively regulate PI3K. Inhibition of miR-429 suppressed low-dose LPS-mediated oxidative stress and inflammation via activation of the PI3K/AKT/Nrf2 pathway. In vivo, miR-429 was detectable in neurons. Inhibition of miR-429 blocked low-dose LPS-mediated oxidative stress and inflammation via activation of the PI3K/AKT/Nrf2 pathway. Overall, low-dose LPS was found to alleviate SCI-induced neuronal oxidative stress and inflammatory response by down-regulating miR-429 to activate the PI3K/AKT/Nrf2 pathway.