Low-dose Lipopolysaccharide Research Articles

Generation of a robust model for inducing autoimmune arthritis in Sprague Dawley rats

IntroductionThe prerequisite for any experimental model in animals is similarity with the human disease, uniformity in disease severity and incidence. In antigen-induced arthritis models it is generally recognized that the major limitation is inconsistency in terms of incidence and severity. As access to strains like DBA/1 mice or Lewis rats is difficult for resource restrained laboratories, this study aimed to establish a robust and reproducible animal model of rheumatoid arthritis (RA). MethodsMultiple approaches were undertaken for inducing arthritis in Sprague Dawley (SD) and Wistar rats using complete Freund's adjuvant (CFA), collagen type II (CII) emulsion, or different combinations of CII with low dose CFA along with lipopolysaccharide (LPS). The development of arthritis was evaluated by measuring paw edema, arthritis score, body weight, splenic index, histopathology and radiography of paw tissues. ResultsThe combination of CII with low dose CFA and one injection of LPS resulted in 100% incidence of arthritis with disease severity ranging from moderate to severe and results were corroborated by histopathology and radiography. DiscussionIn a head-to-head comparison between SD and Wistar rats, the disease profile was better sustained and consistent in SD rats, and the use of CII with low dose CFA and LPS induced features akin to human RA. Taken together, a reproducible model of arthritis was established which can be replicated in any laboratory with limited resources.

Amitriptyline inhibits nonalcoholic steatohepatitis and atherosclerosis induced by high-fat diet and LPS through modulation of sphingolipid metabolism.

We reported previously that increased acid sphingomyelinase (ASMase)-catalyzed hydrolysis of sphingomyelin, which leads to increases in ceramide and sphingosine 1 phosphate (S1P), played a key role in the synergistic upregulation of proinflammatory cytokines by palmitic acid (PA), a major saturated fatty acid, and lipopolysaccharide (LPS) in macrophages. Since macrophages are vital players in nonalcoholic steatohepatitis (NASH) and atherosclerosis, we assessed the effect of ASMase inhibition on NASH and atherosclerosis cooperatively induced by high-PA-containing high-fat diet (HP-HFD) and LPS in LDL receptor-deficient (LDLR-/-) mice. LDLR-/- mice were fed HP-HFD, injected with low dose of LPS and treated with or without the ASMase inhibitor amitriptyline. The neutral sphingomyelinase inhibitor GW4869 was used as control. Metabolic study showed that both amitriptyline and GW4869 reduced glucose, lipids, and insulin resistance. Histological analysis and Oil Red O staining showed that amitriptyline robustly reduced hepatic steatosis while GW4869 had modest effects. Interestingly, immunohistochemical study showed that amitriptyline, but not GW4869, strongly reduced hepatic inflammation. Furthermore, results showed that both amitriptyline and GW4869 attenuated atherosclerosis. To elucidate the underlying mechanisms whereby amitriptyline inhibited both NASH and atherosclerosis, but GW4869 only inhibited atherosclerosis, we found that amitriptyline, but not GW4869, downregulated proinflammatory cytokines in macrophages. Finally, we found that inhibition of sphingosine 1 phosphate production is a potential mechanism whereby amitriptyline inhibited proinflammatory cytokines. Collectively, this study showed that amitriptyline inhibited NASH and atherosclerosis through modulation of sphingolipid metabolism in LDLR-/- mice, indicating that sphingolipid metabolism in macrophages plays a crucial role in the linkage of NASH and atherosclerosis.

Trained immunity modulates inflammation-induced fibrosis

Chronic inflammation and fibrosis can result from inappropriately activated immune responses that are mediated by macrophages. Macrophages can acquire memory-like characteristics in response to antigen exposure. Here, we show the effect of BCG or low-dose LPS stimulation on macrophage phenotype, cytokine production, chromatin and metabolic modifications. Low-dose LPS training alleviates fibrosis and inflammation in a mouse model of systemic sclerosis (SSc), whereas BCG-training exacerbates disease in this model. Adoptive transfer of low-dose LPS-trained or BCG-trained macrophages also has beneficial or harmful effects, respectively. Furthermore, coculture with low-dose LPS trained macrophages reduces the fibro-inflammatory profile of fibroblasts from mice and patients with SSc, indicating that trained immunity might be a phenomenon that can be targeted to treat SSc and other autoimmune and inflammatory fibrotic disorders.

Central and peripheral immune responses to low-dose lipopolysaccharide in a mouse model of the 15q13.3 microdeletion

Carriers of the human 15q13.3 microdeletion (MD) present with a variable spectrum of neuropathological phenotypes that range from asymptomatic to severe clinical outcomes, suggesting an interplay of genetic and non-genetic factors. The most common 2 MB 15q13.3 MD encompasses six genes (MTMR10, FAN1, TRPM1, KLF13, OTUD7A, and CHRNA7), which are expressed in neuronal and non-neuronal tissues. The nicotinic acetylcholine receptor (nAChR) α7, encoded by CHRNA7, is a key player in the cholinergic anti-inflammatory pathway, and the transcription factor KLF13 is also involved in immune responses. Using a mouse model with a heterozygous deletion of the orthologous region of the human 15q13.3 (Df[h15q13]/+), the present study examined peripheral and central innate immune responses to an acute intraperitoneal (i.p.) injection of the bacteriomimetic, lipopolysaccharide (LPS) (100 μg/kg) in adult heterozygous (Het) and wildtype (WT) mice. Serum levels of inflammatory markers were measured 2 h post injection using a Multiplex assay. In control saline injected animals, all measured cytokines were at or below detection limits, whereas LPS significantly increased serum levels of interleukin 1beta (IL-1β), tumor necrosis factor alpha (TNF-α), IL-6 and IL-10, but not interferon-γ. There was no effect of genotype but a sexual dimorphic response for TNF-α, with females exhibiting greater LPS-induced TNF-α serum levels than males. In situ hybridization revealed similar increases in LPS-induced c-fos mRNA expression in the dorsal vagal complex in all groups. The hippocampal expression of the pro-inflammatory cytokines was evaluated by real-time quantitative PCR. LPS-treatment resulted in significantly increased mRNA expression for IL-1β, IL-6, and TNF-α compared to saline controls, with no effect of genotype, but a significant sex-effect was detected for IL-1β. The present study provided no evidence for interactive effects between the heterozygous 15q13.3 MD and a low-dose LPS immune challenge in innate peripheral or central immune responses, although, sex-differential effects in males and females were detected.

"Females Are Not Just 'Protected' Males": Sex-Specific Vulnerabilities in Placenta and Brain after Prenatal Immune Disruption.

Current perceptions of genetic and environmental vulnerabilities in the developing fetus are biased toward male outcomes. An argument is made that males are more vulnerable to gestational complications and neurodevelopmental disorders, the implication being that an understanding of disrupted development in males is sufficient to understand causal mechanisms that are assumed to be similar but attenuated in females. Here we examine this assumption in the context of immune-driven alterations in fetal brain development and related outcomes in female and male mice. Pregnant C57BL/6 mice were treated with low-dose lipopolysaccharide at embryonic day 12.5. Placental pathology, acute fetal brain inflammation and hypoxia, long-term changes in adult cortex cytoarchitecture, altered densities and ratio of excitatory (Satb2+) to inhibitory (parvalbumin+) neuronal subtypes, postnatal growth, and behavior outcomes were compared between male and female offspring. We find that while males experience more pronounced placental pathology, fetal brain hypoxia, depleted PV and Satb2+ densities, and social and learning-related behavioral abnormalities, females exhibit unique acute inflammatory signaling in fetal brain, postnatal growth delay, opposite alterations in cortical PV densities, changes in juvenile behavior, delayed postnatal body growth, and elevated anxiety-related behavior as adults. While males are more severely impacted by prenatal immune disruption by several measures, females exposed to the same insult exhibit a unique set of vulnerabilities and developmental consequences that is not present in males. Our results clearly outline disparate sex-specific features of prenatal vulnerability to inflammatory insults and warn against the casual extrapolation of male disease mechanisms to females.

Locally instructed CXCR4hi neutrophils trigger environment-driven allergic asthma through the release of neutrophil extracellular traps.

Low exposure to microbial products, respiratory viral infections and air pollution is a major risk factor for allergic asthma, yet the mechanistic links between such conditions and host susceptibility to type 2 allergic disorders remain unclear. Through the use of single-cell RNA sequencing (scRNA-seq), we characterized lung neutrophils in mice exposed to a pro-allergic, low dose of lipopolysaccharides (LPSlo) or a protective, high dose of LPS (LPShi) before exposure to house dust mite (HDM). Unlike exposure to LPShi, exposure to LPSlo instructed recruited neutrophils to upregulate the expression of the chemokine receptor CXCR4 and to release neutrophil extracellular traps (NETs). The LPSlo-induced neutrophils and NETs potentiated the uptake of HDM by CD11b+Ly-6C+ dendritic cells (DCs) and type 2 allergic airway inflammation in response to HDM. NETs derived from CXCR4hi neutrophils were also needed to mediate allergic asthma triggered by infection with influenza virus or exposure to ozone. Our study indicates that apparently unrelated environmental risk factors can shape recruited lung neutrophils to promote the initiation of allergic asthma.

Abstract # 3232 Lipopolysaccharide (LPS)-induced tolerance and sensitization: Effects on primary cultures of the afferent nociceptive system

Metabolism and inflammation are linked at many levels. Sickness behaviors are elicited by the immune system’s response to antigenic stimuli, and include changes in feeding and metabolism. The immune system is also regulated by the circadian (daily) clock, which generates endogenous rhythms, and synchronizes these rhythms to the light-dark cycle. Modern society has resulted in chronic misalignment or desynchronization of the circadian clock and the external environment. We have demonstrated that circadian desynchronization (CD) in mice alters metabolic function, and also affects both peripheral and central immune responses following a low-dose lipopolysaccharide (LPS) challenge. However, it is unclear how this altered immune response impacts sickness behaviors and metabolism following challenge. To test this, we housed male mice in circadian desynchronized (10-hours light:10-hours dark) or control (12-hours light:12-hours dark) conditions for 5–6 weeks. We then challenged mice with LPS (i.p., 0.4 mg/kg) or PBS and measured changes in body mass, feeding, drinking and locomotion using a comprehensive phenotyping system. Plasma, liver, and brain were collected 36 h post-inoculation (hpi) and inflammatory messengers were measured via multiplex cytokine/chemokine array and qPCR. We find that recovery of locomotion and body mass is prolonged in CD mice following LPS challenge. Additionally, at 36 hpi the expression of several proinflammatory cytokines differ depending on pre-inoculation lighting conditions. Our findings add to the growing literature which documents how desynchronization of circadian rhythms can lead to disrupted immune responses and changes in metabolic function.

Systemic LPS-induced neuroinflammation increases the susceptibility for proteasome inhibition-induced degeneration of the nigrostriatal pathway

IntroductionBesides proteasome dysfunction, neuroinflammation is a common feature in the pathogenesis of Parkinson's disease (PD). Accordingly, peripheral inflammation has been shown to increase the susceptibility of the brain for nigrostriatal degeneration by inducing activation of glial cells and release of pro-inflammatory cytokines in the brain. Given that current animal models of PD fail to recapitulate the pathophysiology occurring in idiopathic PD, the aim of this study was to combine two pathogenic mechanisms (i.e. neuroinflammation and proteasome inhibition) to create a dual-hit mouse model of PD. MethodsWe repeatedly injected mice with a low dose of LPS (250 μg/kg/day i. p. for four days) to induce neuroinflammation, followed by a unilateral intranigral injection of lactacystin (LAC; 3 μg). Seven days later, mice were evaluated behaviorally to assess locomotion, anxiety- and depressive-like behavior. Nigrostriatal degeneration was analyzed by measuring striatal dopamine loss as well as loss of nigral dopaminergic neurons. Neuroinflammation was confirmed by quantifying microglial cells in the substantia nigra (SN) and cytokine expression in the striatum. ResultsRepeated systemic LPS injections increase the number of microglial cells in the SN and induce a mixed profile of pro- and anti-inflammatory cytokines in the striatum without affecting the integrity of the nigrostriatal pathway. Systemic LPS-induced neuroinflammation, however, increases the susceptibility of the nigrostriatal pathway for LAC-induced degeneration. ConclusionRecapitulating two relevant etiopathogenic mechanisms of PD - neuroinflammation and proteasome inhibition-, we propose this dual-hit model as a relevant mouse model for PD that could be used to investigate potential therapeutic targets.

Genetic lines respond uniquely within the chicken thymic transcriptome to acute heat stress and low dose lipopolysaccharide

Exposure to high temperatures is known to impair immune functions and disease resistance of poultry. Characterizing changes in the transcriptome can help identify mechanisms by which immune tissues, such as the thymus, respond to heat stress. In this study, 22-day-old chickens from two genetic lines (a relatively resistant Fayoumi line and a more susceptible broiler line) were exposed to acute heat stress (35 °C) and/or immune simulation with lipopolysaccharide (LPS; 100 µg/kg). Transcriptome responses in the thymus were identified by RNA-sequencing (RNA-seq). Expression of most genes was unaffected by heat and/or LPS in the Fayoumi line, whereas these treatments had more impact in the broiler line. Comparisons between the broiler and Fayoumi transcriptomes identified a large number of significant genes both at homeostasis and in response to treatment. Functional analyses predicted that gene expression changes impact immune responses, apoptosis, cell activation, migration, and adhesion. In broilers, acute heat stress changed thymic expression responses to LPS and could impact thymocyte survival and trafficking, and thereby contribute to the negative effects of high temperatures on immune responses. Identification of these genes and pathways provides a foundation for testing targets to improve disease resistance in heat-stressed chickens.

Transcription analysis of the response of the porcine adrenal cortex to a single subclinical dose of lipopolysaccharide from Salmonella Enteritidis

Lipopolysaccharide (LPS) is a bacterial endotoxin which can participate in the induction of inflammatory responses. LPS may also play a significant role in some neurodegenerative, oncological and metabolic disorders. The aim of the current study was to determine the effect of a subclinical low single dose of LPS from Salmonella Enteritidis administrated in vivo on the transcriptome of porcine adrenal cortex cells, especially gene expression levels, long non-coding RNA (lncRNA) profiles, alternative splicing events and RNA editing sites using RNA-seq technology. The subclinical dose of LPS changed the expression of 354 genes, 27 lncRNA loci and other unclassified RNAs. An analysis of alternative splicing events revealed 104 genes with differentially expressed splice junction sites, and the single nucleotide variant calling approach supported the identification of 376 canonical RNA editing candidates and 7249 allele-specific expression variants. The obtained results suggest that the RIG-I-like receptor signaling pathway, may play a more important role than the Toll-like signaling pathway after the administration of a subclinical dose of LPS. Single subclinical dose of LPS can affect the expression profiles of genes coding peptide hormones, steroidogenic enzymes and transcriptional factors, and modulate the endocrine functions of the gland.

Perinatal Breathing Patterns and Survival in Mice Born Prematurely and at Term.

Infants born prematurely, often associated with maternal infection, frequently exhibit breathing instabilities that require resuscitation. We hypothesized that breathing patterns during the first hour of life would be predictive of survival in an animal model of prematurity. Using plethysmography, we measured breathing patterns during the first hour after birth in mice born at term (Term 19.5), delivered prematurely on gestational day 18.5 following administration of low-dose lipopolysaccharide (LPS; 0.14 mg/kg) to pregnant dams (LPS 18.5), or delivered on gestational day 18.7 or 17.5 by caesarian section (C-S 18.5 and C-S 17.5, respectively). Our experimental approach allowed us to dissociate effects caused by inflammation, from effects due to premature birth in the absence of an inflammatory response. C-S 17.5 mice did not survive, whereas mortality was not increased in C-S 18.5 mice. However, in premature pups born at the same gestational age (day 18.5) in response to maternal LPS injection, mortality was significantly increased. Overall, mice that survived had higher birth weights and showed eupneic or gasping activity that was able to transition to normal breathing. Some mice also exhibited a “saw tooth” breathing pattern that was able to transition into eupnea during the first hour of life. In contrast, mice that did not survive showed distinct, large amplitude, long-lasting breaths that occurred at low frequency and did not transition into eupnea. This breathing pattern was only observed during the first hour of life and was more prevalent in LPS 18.5 and C-S 18.5 mice. Indeed, breath tidal volumes were higher in inflammation-induced premature pups than in pups delivered via C-section at equivalent gestational ages, whereas breathing frequencies were low in both LPS-induced and C-section-induced premature pups. We conclude that a breathing pattern characterized by low frequency and large tidal volume is a predictor for the failure to survive, and that these characteristics are more often seen when prematurity occurs in the context of maternal inflammation. Further insights into the mechanisms that generate these breathing patterns and how they transition to normal breathing may facilitate development of novel strategies to manage premature birth in humans.

Antagonism of major histocompatibility complex class II invariant chain peptide during chronic lipopolysaccharide treatment rescues autoregulatory behavior.

Toll-like receptor 4 (TLR4) activation contributes to vascular dysfunction in pathological conditions such as hypertension and diabetes, but the role of chronic TLR4 activation on renal autoregulatory behavior is unknown. We hypothesized that subclinical TLR4 stimulation with low-dose lipopolysaccharide (LPS) infusion increases TLR4 activation and blunts renal autoregulatory behavior. We assessed afferent arteriolar autoregulatory behavior in male Sprague-Dawley rats after prolonged LPS (0.1 mg·kg-1·day-1 sq) infusion via osmotic minipump for 8 or 14 days. Some rats also received daily cotreatment with either anti-TLR4 antibody (1 μg ip), competitive antagonist peptide (CAP; 3 mg/kg ip) or tempol (2 mmol/l, drinking water) throughout the 8-day LPS treatment period. Autoregulatory behavior was assessed using the in vitro blood-perfused juxtamedullary nephron preparation. Selected physiological measures, systolic blood pressure and baseline diameters were normal and similar across groups. Pressure-dependent vasoconstriction averaged 72 ± 2% of baseline in sham rats, indicating intact autoregulatory behavior. Eight-day LPS-treated rats exhibited significantly impaired pressure-mediated vasoconstriction (96 ± 1% of baseline), whereas it was preserved in rats that received anti-TLR4 antibody (75 ± 3%), CAP (84 ± 2%), or tempol (82 ± 2%). Using a 14-day LPS (0.1 mg·kg-1·day-1 sq) intervention protocol, CAP treatment started on day 7, where autoregulatory behavior is already impaired. Systolic blood pressures were normal across all treatment groups. Fourteen-day LPS treatment retained the autoregulatory impairment (95 ± 2% of baseline). CAP intervention starting on day 7 rescued pressure-mediated vasoconstriction with diameters decreasing to 85 ± 1% of baseline. These data demonstrate that chronic subclinical TLR4 activation impairs afferent arteriolar autoregulatory behavior through mechanisms involving reactive oxygen species and major histocompatibility complex class II activation.

Cortisol, progesterone, 17α-hydroxyprogesterone, and TSH responses in dogs injected with low-dose lipopolysaccharide.

BackgroundStress and diseases such as endotoxemia induce cortisol synthesis through a complex biosynthetic pathway involving intermediates (progesterone, and 17α-hydroxyprogesterone (17α-OHP)) and suppression of the hypothalamus-pituitary-thyroid axis.ObjectiveTo measure plasma concentrations of cortisol, progesterone, 17α-OHP, and thyroid stimulating hormone (TSH) in dogs experimentally injected with intravenous low-dose lipopolysaccharide (LPS). Our hypothesis was that LPS treatment would elicit a significant increase in cortisol and its precursors, and a significant decrease in TSH concentration.MethodsHormone measurements were performed on blood samples left over from a previous investigation (2011) on the effect of low-dose LPS on hematological measurands. Five sexually intact female dogs, none in estrous at the time of the study, were administered saline treatment two weeks prior to LPS treatment. LPS was administered intravenously at a dose of 0.1 µg/kg. Blood was collected before (baseline, time -24 hours) and 3-, 6- and 24-hours post-injection. Mixed model analysis for repeated measures was used, with both treatment and time as the repeated factors. Ranked transformation were applied when diagnostic analysis exhibited violation of normality and equal variance assumptions. Post hoc multiple comparisons were performed with Tukey’s adjustment. Statistical significance was defined as p < 0.05.ResultsSignificant differences relative to baseline values were detected following both treatments. Compared to baseline, dogs had significantly higher cortisol and 17α-OHP at 3-hours, and significantly lower TSH at 3- and 6-hours following LPS treatment. Dogs had significantly lower TSH at 6- and 24- following saline treatment. Though not statistically significant, the trend in progesterone concentrations was similar to cortisol and 17α-OHP, with an increase at 3-hours post-injection followed by a decrease close to baseline following both LPS and saline. Cortisol and 17α-OHP concentrations were higher after LPS treatment than after saline treatment at 3- and 6-hours post-injection, but differences were not statistically significant, and no significant differences between treatments were detected for any other hormone or timepoint.Discussion and conclusionCortisol and its adrenal precursors are released in the bloodstream following a low dose of LPS, while TSH appears to decrease. Similar changes occurred following saline treatment, suggesting that even routine handling and saline injection in conditioned dogs can elicit alterations in the internal equilibrium with subsequent modification of both hypothalamus-pituitary-adrenal and thyroid axes. Changes to adrenal and thyroid hormone concentrations must be interpreted in light of clinical information. Further studies are needed to elucidate mechanisms of adrenal steroidal hormone synthesis and secretion in response to various stressful stimuli in both neutered and intact animals.

Usefulness of Monocytes/macrophages Activated With Low-dose Lipopolysaccharide in Tumor Tissue and Adipose Tissue of Obesity.

Chronic inflammation is involved in the development of cancer, lifestyle-related diseases, and autoimmune diseases. It also influences the severity of these diseases. Macrophages that accumulate in tumor tissues and adipose tissues of obesity have been shown to increase expression of inflammatory cytokines, thereby inducing inflammatory changes in these tissues. The macrophage phenotype is believed to be important in mediating inflammatory changes in tissues. Recently, monocytes/macrophages activated with low-dose lipopolysaccharide (LPS) were demonstrated to suppress increased expression of monocyte chemotactic protein (MCP)-1 and inflammatory cytokines (interleukin (IL)-1 β, IL-8, and tumor necrosis factor (TNF)-α). By suppressing the increased expression of chemotaxis-related and inflammation-related factors, monocytes/macrophages activated with low-dose LPS are considered to suppress the migration of macrophages into tissues and to regulate inflammatory changes in these tissues, respectively. The effects of macrophages activated with low-dose LPS were different from those of macrophages activated with high-dose LPS. In this review, we discuss the usefulness of monocytes/macrophages activation by low-dose LPS.

SUN-171 PRETREATMENT WITH LOW DOSE LIPOPOLYSACCHARIDE PREVENTS ISCHEMIA-REPERFUSION INDUCED RENAL VASCULAR CONGESTION IN WKY RATS.

Our lab has previously shown red blood cell (RBC) congestion in the renal outer medulla is critical in determining the recovery of renal function following ischemia-reperfusion (IR). While pre-conditioning with lipopolysaccharide (LPS) has been reported to provide protection against IR-induced tubular injury, the effect of low dose LPS on renal medullary congestion has not been directly examined. We hypothesized prior exposure to low dose LPS would induce mild inflammation, promote rouleaux and increase RBC congestion following IR. Study 1: LPS dose response in 10wk old male Wistar-kyoto rats (WKY). Rats were injected (i.p.) with either 10 (n=3), 100 (n=3), 1000 (n=6) ug/kg LPS or saline (n=5) once daily for 7 days prior to IR. Low dose LPS concentrations are less than 1/10th of the dose reported to cause hemodynamic dysfunction. Study 2: Preconditioning vs acute low dose LPS in male WKY. Rats were treated with either 1) 1 injection (i.p) of LPS (1000ug/kg, n=3) or saline (n=3) 24h prior to IR, 2) daily injection of LPS (1000ug/kg, n=4) or saline (n=4) for 3 days prior to IR, or 3) daily injections of LPS (1000ug/kg, n=5) or saline (n=4) for 7 days prior to IR. Following pretreatment, all animals were anesthetized for 30min (Study 1) or 45min (Study 2) ischemia with 24h reperfusion, and humanely sacrificed. Kidneys were collected for histological analysis of RBC congestion (blinded scoring scale 0-5) and tubular injury (blinded, 0-100%). Blood was collected at baseline, pre-IR, and sacrifice for measurement of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), blood urea nitrogen (BUN), and serum creatinine (SCr). Study 1: CRP and ESR measurements indicated modest inflammation and rouleaux formation in LPS (1000 ug/kg) pretreated animals (ESR: 5.9mm±1.2) compared to control (ESR: 3.2mm±1.4). Most striking, medullary peritubular congestion showed LPS pretreatment dose dependently attenuated congestion when compared to saline treated controls (Fig. 1). Study 2: Preconditioning with low dose LPS (1 injection daily for 3 or 7days) induced mild inflammation and reduced RBC congestion in the renal medulla (7 day congestion score: LPS 1.3±0.94, control 3.5±0.54). In these same animals, tubular injury showed no difference between LPS and control groups (p= 0.397). In contrast, a single, acute injection of low dose LPS 24h prior to IR demonstrated similar renal medullary RBC congestion as saline injected animals (Congestion score: LPS 3.2±0.83, control 2.2±1.09). We conclude that despite promoting inflammation, and rouleaux, 3-7days of pretreatment with low dose LPS attenuates RBC congestion in the renal medulla following IR. The effect appears to be largely independent of tubular injury, suggesting the phenomenon is not secondary to reduced parenchymal damage. As sustained congestion and hypoxia following IR may lead to progressive renal injury in the medulla after renal blood flow and GFR returns towards normal, understanding the mechanisms by which low dose LPS prevents renal congestion may lead to novel therapeutic approaches to improve recovery from ischemic acute kidney injury.

Abstract 4311: Mechanisms responsible for the inflammatory polarization of monocytes by subclinical low-dose LPS

Abstract Dynamic polarizations of innate leukocytes such as monocytes and macrophages have been increasingly recognized to play key roles in the pathogenesis and/or treatment of human disease. However, mechanisms responsible for distinct polarizations of monocytes are not well understood. Our previous studies indicate that monocytes can be persistently polarized into a low-grade inflammatory state by subclinical super-low dose LPS (bacterial lipopolysaccharide). In this current study, we further characterized the unique polarization of monocytes by super-low dose LPS and its underlying mechanisms. We observed that super-low dose LPS preferentially induced the expression of inflammatory mediators such as MCP-1 while potently suppressed the expression of homeostatic mediators such as Catalase and FPN. Super-low dose LPS activated the inflammatory signaling molecules such as p38, CamKII, and NFkB. In contrast, super-low dose LPS suppressed the basal activation of immune suppressing molecules such as AMPK and SIRT3. Taken together, our data reveal novel insights regarding the persistent polarization of inflammatory monocyte, an emerging key player in various human diseases including cancer. Citation Format: Kisha Pradhan. Mechanisms responsible for the inflammatory polarization of monocytes by subclinical low-dose LPS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4311.

The IRE1 signaling pathway is involved in the protective effect of low-dose LPS on myocardial ischemia-reperfusion injury

AimThe IRE1 signaling pathway is implicated in I/R injury. However, little is known about the involvement of this pathway in low-dose LPS treatment of myocardial I/R injury. Thus, an attempt was made to determine the relationship between the IRE1 pathway and I/R injury using rats or in vitro H9C2 cell myocardial I/R injury models. Main methodsSprague-Dawley rats and cultured H9C2 cells were pretreated with low-dose LPS and subjected to myocardial I/R injury models. Key findingsLow-dose LPS did not affect normal rat or cellular function. Compared with the I/R group, treatment with LPS attenuated myocardial apoptosis, decreased plasma LDH and CK-MB activities, reduced myocardium infarct size, and downregulated caspase-3 expression. Moreover, the protein or mRNA expression levels of the IRE1 signaling pathway-related proteins Grp78, IRE1, p-ASK1, ASK1, p-JNK, and JNK were notably increased during I/R injury but significantly decreased by low-dose LPS treatment both in rats and in H9C2 cells. SignificanceLow-dose LPS exhibited therapeutic effects in myocardial I/R injury. Most importantly, the cardioprotective mechanism of low-dose LPS may be associated with the IRE1 signaling pathway.

Exogenous Administration of Low-Dose Lipopolysaccharide Potentiates Liver Fibrosis in a Choline-Deficient l-Amino-Acid-Defined Diet-Induced Murine Steatohepatitis Model

Various rodent models have been proposed for basic research; however, the pathogenesis of human nonalcoholic steatohepatitis (NASH) is difficult to closely mimic. Lipopolysaccharide (LPS) has been reported to play a pivotal role in fibrosis development during NASH progression via activation of toll-like receptor 4 (TLR4) signaling. This study aimed to clarify the impact of low-dose LPS challenge on NASH pathological progression and to establish a novel murine NASH model. C57BL/6J mice were fed a choline-deficient l-amino-acid-defined (CDAA) diet to induce NASH, and low-dose LPS (0.5 mg/kg) was intraperitoneally injected thrice a week. CDAA-fed mice showed hepatic CD14 overexpression, and low-dose LPS challenge enhanced TLR4/NF-κB signaling activation in the liver of CDAA-fed mice. LPS challenge potentiated CDAA-diet-mediated insulin resistance, hepatic steatosis with upregulated lipogenic genes, and F4/80-positive macrophage infiltration with increased proinflammatory cytokines. It is noteworthy that LPS administration extensively boosted pericellular fibrosis with the activation of hepatic stellate cells in CDAA-fed mice. Exogenous LPS administration exacerbated pericellular fibrosis in CDAA-mediated steatohepatitis in mice. These findings suggest a key role for LPS/TLR4 signaling in NASH progression, and the authors therefore propose this as a suitable model to mimic human NASH.

1889-P: Inhibition of Acid Sphingomyelinase Attenuates Nonalcoholic Steatohepatitis and Atherosclerosis Induced by High-Fat Diet and LPS in LDL Receptor-Deficient Mice

Macrophage-mediated inflammation plays an essential role in diabetes-associated nonalcoholic steatohepatitis (NASH) and atherosclerosis. Our previous study has shown that acid sphingomyelinase (ASMase) plays a key role in the synergistic stimulation of inflammatory signaling by saturated fatty acid palmitate and lipopolysaccharide (LPS) in macrophages. In the current study, we assessed the effect of ASMase inhibition on NASH and atherosclerosis induced by lard-based, high palmitate-containing high-fat diet (HP-HFD) in combination with LPS in LDL receptor-knockout (LDLR-/-) mice. LDLR-/- mice were fed HP-HFD for 20 weeks and treated with or without a low dose of LPS and amitriptyline, an inhibitor of ASMase. Metabolic assays showed that amitriptyline reduced bodyweight, lipids and insulin resistance induced by HP-HFD or HP-HFD plus LPS. Histological study and Oil Red O staining of livers showed that while mice fed HP-HFD without LPS treatment had hepatic steatosis and hepatocellular ballooning, LPS further increased steatosis and ballooning by 70%. However, amitriptyline attenuated the effect of HP-HFD or HP-HFD plus LPS by 66%. Immunohistochemistry study showed that while mice fed HP-HFD without LPS treatment had a marked immunostaining of F4/80, a biomarker for macrophages, LPS further enhanced F4/80 immunostaining. However, amitriptyline significantly reduced F4/80 immunostaining. Furthermore, results showed that amitriptyline also attenuated atherosclerosis induced by HP-HFD plus LPS. To elucidate the underlying mechanisms, we found that amitriptyline downregulated proinflammatory cytokines by potently reducing sphingosine 1 phosphate, a bioactive sphingolipid. Taken together, this study showed that ASMase inhibition attenuated both NASH and atherogenesis induced by HP-HFD plus LPS and one of the mechanisms potentially involved in the attenuation is inhibition of S1P production. Disclosure Z. Lu: None. Y. Li: None. M.F. Lopes-Virella: None. Y. Huang: None. Funding U.S. Department of Veterans Affairs (1I01CX001825-01); National Institutes of Health

Peroxiredoxin II negatively regulates BMP2-induced osteoblast differentiation and bone formation via PP2A C\u03b1-mediated Smad1/5/9 dephosphorylation

Peroxiredoxin II (Prx II), an antioxidant enzyme in the Prx family, reduces oxidative stress by decreasing the intracellular ROS levels. Osteoblast differentiation is promoted by bone morphogenetic protein 2 (BMP2), which upregulates the expression of osteoblast differentiation marker genes, through Smad1/5/9 phosphorylation. We found that Prx II expression was increased by a high dose of lipopolysaccharide (LPS) but was not increased by a low dose of LPS. Prx II itself caused a decrease in the osteogenic gene expression, alkaline phosphatase (ALP) activity, and Smad1/5/9 phosphorylation induced by BMP2. In addition, BMP2-induced osteogenic gene expression and ALP activity were higher in Prx II knockout (KO) cells than they were in wild-type (WT) cells. These inhibitory effects were mediated by protein phosphatase 2A Cα (PP2A Cα), which was increased and is known to induce the dephosphorylation of Smad1/5/9. The overexpression of Prx II increased the expression of PP2A Cα, and PP2A Cα was not expressed in Prx II KO cells. Moreover, PP2A Cα reduced the level of BMP2-induced osteogenic gene expression and Smad1/5/9 phosphorylation. LPS inhibited BMP2-induced Smad1/5/9 phosphorylation and the suppressed phosphorylation was restored by the PP2A inhibitor okadaic acid (OA). Bone phenotype analyses using microcomputed tomography (μCT) revealed that the Prx II KO mice had higher levels of bone mass than the levels of the WT mice. We hypothesize that Prx II has a negative role in osteoblast differentiation through the PP2A-dependent dephosphorylation of Smad1/5/9.