Dose Of LPS Research Articles

Sex-Differences Influence Depressive-Like Behaviour via Alterations in Microglial Expression of GIF-1, TREM2, and IL-1β in an Acute Lipopolysaccharide-Induced Murine Neuroinflammation Model

ABSTRACT Background Neurodegenerative diseases (NDs) have caused serious health issues worldwide. A growing body of evidence suggests a correlation between neuroinflammation and abnormal microglial activity with ND symptoms. Microglia survey play crucial roles in CNS during health and the injury. It is proposed that sex affects microglial roles during inflammation, resulting in mouse behavioural changes and expression alterations in key markers related to microglia functions. Methods Male and female C57BL/6 mice were injected with a single dose of LPS (5 mg/kg, i.p.) or saline. After 48 h, an open field test was conducted, followed by brain tissues collection for measuring the expression of IGF-1, IL-1β and TREM2 and Immunohistochemistry (IHC) analysis for NLRP3 level. Results Males displayed greater depressive-like behaviour in the OFT, with lower levels of IGF-1, IL-1β, and NLRP3 and high TREM2 expression. Female mice did not exhibit this behaviour, in contrast to male mice, they exhibited increased IL-1β and NLRP3 expression Discussion This study revealed that LPS-induced sex-specific changes in genes involved in neuronal cell survival caused behavioural alterations in male mice. Moreover, females had observed inflammatory responses that had no impact on behavioural alterations. Overall, both sexes exhibited sex-specific microglial activation states.

Splenic γδ T cells mediate antidepressant and prophylactic actions of arketamine in lipopolysaccharide-induced depression in mice

Arketamine, the (R)-enantiomer of ketamine, exhibits both therapeutic and sustained prophylactic effects in an inflammation-driven model of depression, although the precise mechanisms remain elusive. Given the involvement of γδ T cells in inflammatory processes, this study explored their role in the effects of arketamine. To assess therapeutic outcomes, mice received lipopolysaccharide (LPS:1.0 mg/kg), followed by either arketamine (10 mg/kg) or saline. For prophylactic assessment, arketamine or saline was administered six days prior to LPS exposure. A single dose of LPS (1.0 mg/kg) reduced the proportion of γδ T cells in the spleen but did not affect their levels in the blood, prefrontal cortex, or small intestine. Arketamine mitigated LPS-induced splenomegaly, counteracted the elevation of plasma interleukin-6 levels and the reduction in the proportion of splenic γδ T cells, and alleviated depression-like behavior as assessed by the forced swimming test. Notably, negative correlations were observed between the proportion of splenic γδ T cells and indicators of inflammation and depression. Furthermore, pretreatment with a γδ TCR antibody significantly countered the therapeutic and prophylactic effects of arketamine on LPS-induced changes. These findings highlight a novel role for splenic γδ T cells in inflammation-associated depression and suggest the potential of arketamine as a treatment option. Consequently, γδ T cells may represent a novel therapeutic target for inflammation-related depression. Further studies on the role of γδ T cells in depressed patients with inflammation are warranted.

Peripheral Lysosomal Positioning in Inflamed Odontoblasts Facilitates Mineralization.

Odontoblasts, terminally differentiated dentin-producing cells, critically rely on lysosomal functions for intracellular recycling and renewal. Beyond their traditional degradative role, lysosomes actively orchestrate cellular responses to external stimuli through precise and rapid intracellular trafficking and positioning. This study aimed to explore the influence of lysosomal positioning on odontoblast mineralization and the underlying mechanisms implicated in carious inflammation. Human dental pulp stem cells (hDPSCs) were induced to differentiate into human odontoblast-like cells (hOBLCs). hOBLCs were treated with various doses of LPS (0.1, 1, 5 μg/mL) to mimic carious inflammation. Lysosomal positioning was examined by immunofluorescence staining of LAMP1 in healthy and carious human teeth, LPS-treated hOBLCs, mouse lower incisors at postnatal day 2.5, and mineralization medium cultured hDPSCs. Lysosomal positioning was manipulated by knockdown or overexpression of SNAPIN or ARL8B. Mineralization was assessed by ARS staining and expression of DSPP and DMP1. Lysosomal exocytosis was examined by detection of lysosomal-plasma membrane fusion, surface exposure of LAMP1 luminal epitopes (1D4B) and extracellularly released lysosomal enzymes. Peripheral lysosomal positioning was markedly increased in odontoblasts within moderate and extensive carious lesions (P < .001) and in hOBLCs following LPS treatment. Increased peripheral dispersion of lysosomes was similarly observed during odontoblastic differentiation in vivo and in vitro. Moreover, peripheral lysosomal positioning promoted mineralization in inflamed hOBLCs, potentially via mTORC1 signaling pathway and lysosomal exocytosis. Inflammatory stimuli prompted a relocation of lysosomes in odontoblasts, redistributing them from perinuclear location towards the cell periphery, which in turn facilitated mineralization, potentially via mTORC1 signaling and lysosomal exocytosis.

Direct IL-6 inhibition prevents arterial thrombosis by reducing collagen-mediated platelets activation

Abstract Introduction Interleukin 6 (IL-6) has a pivotal role in atherothrombosis. Yet, targeting IL-6 to prevent atherothrombotic events still requires validation of efficacy and safety. The recent phase 2 RESCUE trial reported that direct inhibition of IL-6 ligand by the monoclonal antibody (mAb) Ziltivekimab is safe in patients with elevated cardiovascular risk. Purpose This project investigated the effects of direct IL-6 inhibition on arterial thrombosis, and assessed the underlying cellular mechanisms. Methods Three month old C56Bl/6 male mice received very-low dose LPS i.p. for 4 weeks. During the last week they were randomized to receive in addition either anti-mouse IL-6 mAb 200 μg i.p. every other day or IgG1 isotype control. Then thrombosis of left common carotid artery (LCCA) was induced by laser injury under anesthesia. Platelets of treated mice were isolated and stimulated with either adenosine di-phosphate (ADP), collagen-related peptide (CRP) or thrombin receptor activating peptide (TRAP). Platelets activation was measured by flow-cytometry, as expression of P-selectin and JON/A.The effect of direct IL-6 inhibition was confirmed in patients with stable coronary artery disease (sCAD) by ex-vivo incubation of isolated platelets with either anti-human IL-6 mAb 1.5 μg/mL or IgG1 isotype control. Platelet activation was measured by flow-cytometry, as expression of P-selectin. Results Mice treated with anti-IL6 antibody displayed a significantly longer time-to-occlusion after the LCCA (Figure 1), without any significant difference in coagulation parameters. After stimulation with CRP, platelets were significantly activated in control mice, but not in mice treated with anti-IL6 mAb. Similarly, activation of isolated platelets from patients with sCAD was significantly reduced (Figure 2) by the ex-vivo treatment with anti-IL6 mAb. Conclusion The direct inhibition of IL-6 reduces platelets reactivity to collagen, thereby blunting arterial thrombosis upon endothelial damage. These results provide a potential mechanism to explain the reduction of cardiovascular risk associated to direct IL-6 inhibition. Conclusion The direct inhibition of IL-6 reduces platelets reactivity to collagen, thereby blunting arterial thrombosis upon endothelial damage. These results provide a potential mechanism to explain the reduction of cardiovascular risk associated to direct IL-6 inhibition.Figure 1Figure 2

5114 Establishment of Thyroid Storm Mouse Model and Therapeutic Effects of Ghrelin

Abstract Disclosure: C. Kurimoto: None. Y. Furukawa: None. T. Akamizu: None. A. Doi: None. K. Takeshima: None. S. Morita: None. H. Iwakura: None. H. Ariyasu: None. H. Furuta: None. M. Nishi: None. T. Matsuoka: None. Context: Thyroid storm (TS), a life-threatening condition that can damage multiple organs, is caused by a combination of thyrotoxicosis and severe physical stresses. Infection is the most common triggering factor. Hypercytokinemia is a suggested background, but the pathological condition is unclear and there are no appropriate animal models. Further, TS has limited therapeutic options. Objectives: We aimed to confirm the hypercytokinemia in patients with TS, establish a TS model mouse by triiodothyronine and lipopolysaccharide administration, and explore the therapeutic effects of ghrelin on TS. Methods: We evaluated serum IL-6 levels as a representative marker of hypercytokinemia in patients with TS. As a TS model, C57BL/6 mice were titrated with triiodothyronine and lipopolysaccharide to develop a lethal model with approximately 30% survival at 24 hours. We assessed the survival ratio, mouse sepsis scores and blood biomarkers (IL-6, metanephrine, alanine aminotransferase) and evaluated the effects of ghrelin on these parameters. Results: Serum IL-6 was increased in patients with TS compared with those with Graves' disease without TS (55.7 ± 69.0 vs. 3.13 ± 1.41 pg/mL, P &amp;lt; .05, n=4 each). Next, we titrated the doses of T3 and LPS, aiming to achieve a survival rate of approximately 30% at 24 hours after administration. Two out of five mice in the T3 3.0 mg/kg group died, while none of the eight mice in the T3 1.0 mg/kg group died. Therefore, we determined that the appropriate T3 dose for the TS mouse model is 1.0 mg/kg, which is not lethal when administered alone. In the T3 1.0 mg/kg group (n=8), serum FT3 levels were approximately three times higher (15.3 ± 4.5 vs. 4.5 ± 0.7pg/ml) than those in the control group (n=8). Next, different doses of LPS was added to the T3 group. Survival at 24 hours post-dose was 60% in the T3 + LPS 0.2 mg/kg group (n=5) and 33% in the T3 + LPS 0.5 mg/kg group (n=5), while survival was 100% in the LPS 0.5 mg/kg alone group (n=5),. Based on these findings, the dosage for the murine TS model was determined to be “triiodothyronine 1.0 mg/kg and lipopolysaccharide 0.5 mg/kg”. This TS model group had increased mouse sepsis score, serum IL-6, metanephrine and alanine aminotransferase. In this model, the ghrelin at 300 µg/kg improved the survival rate to 66.7% (P &amp;lt; .01, vs. 0% [saline-treated group]) as well as the mouse sepsis score, and it decreased the serum IL-6 and metanephrine. Conclusion: We confirmed the hypercytokinemia in patients with TS. Next, we established a lethal model simulating TS by inducing “thyrotoxicosis” by exogenous administration of thyroid hormones, and by inducing “sepsis” by intraperitoneal administration of LPS. The model mice exhibited similar pathophysiological status to human TS associated with induction of serum IL-6 and other biomarkers, which were improved by ghrelin. Presentation: 6/1/2024

In Vitro Study of the Differential Anti-Inflammatory Activity of Dietary Phytochemicals upon Human Macrophage-like Cells as a Previous Step for Dietary Intervention.

Chronic inflammatory diseases pose a substantial health challenge globally, significantly contributing to morbidity and mortality. Addressing this issue requires the use of effective anti-inflammatory strategies with fewer side effects than those provoked by currently used drugs. In this study, a range of phytochemicals (phenolic di-caffeoylquinic acid (Di-CQA), flavonoid cyanidin-3,5-diglucoside (Cy3,5DiG), aromatic isothiocyanate sinalbin (SNB) and aliphatic isothiocyanate sulforaphane (SFN)) sourced from vegetables and fruits underwent assessment for their potential anti-inflammatory activity. An in vitro model of human macrophage-like cells treated with a low dose of LPS to obtain a low degree of inflammation that emulates a chronic inflammation scenario revealed promising results. Cell viability and production of the key pro-inflammatory cytokines were assessed in the presence of various phytochemicals. The compounds Di-CQA and Cy-3,5-DiG, within low physiologically relevant doses, demonstrated notable anti-inflammatory effects by significantly reducing the production of key pro-inflammatory cytokines TNF-α and IL-6 without affecting cell viability. These findings underscore the potential of plant-derived bioactive compounds as valuable contributors to the prevention or treatment of chronic inflammatory diseases. These results suggest that these compounds, whether used individually or as part of natural mixtures, hold promise for their inclusion in nutritional interventions designed to mitigate inflammation in associated pathologies.

Lie to me to lay with me: Females deceive males via terminal investment.

Historically, males have frequently been portrayed as the manipulative and deceptive gender, while females are often seen as adopting a coy and passive role. In this context, it is proposed that males use a terminal investment strategy, misleading females about their true poor condition, while females passively opt to mate with these deceptive males. However, we hypothesize that females in suboptimal condition may also engage in a terminal investment strategy by mimicking or enhancing their attractiveness to match that of females in better conditions. We studied this hypothesis in Tenebrio molitor, by subjecting females to three varying doses of lipopolysaccharides of Escherichia coli (LPS; 0.25, 0.5, or 1 mg ml-1), or three doses of the pro-oxidant Paraquat (PQ; 20, 40 or 80 mM), and subsequently assessing their survival and attractiveness to males. The LPS treatments and 20 mM of PQ had no significant effect on the survival or attractiveness of the females. However, females treated with 40 or 80 mM PQ survived fewer days compared to the control group. Those injected with 40 mM were more attractive than their control counterparts, while those treated with 80 mM were less attractive. Since the identical doses of LPS, which induce terminal investment in males, had no effect on females, we suggest sexual dimorphism in terminal investment. Furthermore, similar to males, if the stressor reaches a sufficiently high level, the signal becomes honest. These findings highlight how the quantity of stressors influences support for the terminal investment strategy in both males and females. Notably, this study challenges prevailing notions regarding gender roles in sexual selection, indicating that females, not just males, conceal their poor condition to attract mating partners.

Acute Postnatal Inflammation Alters Adult Microglial Responses to LPS that Are Sex-, Region- and Timing of Postnatal Inflammation-Dependent.

Adverse events in early life can have impact lasting into adulthood. We investigated the long-term effects of systemic inflammation during postnatal development on adult microglial responses to LPS in two CNS regions (cortex, cervical spinal cord) in male and female rats. Inflammation was induced in Sprague-Dawley rats by lipopolysaccharide (LPS, 1 mg/kg) administered intraperitoneally during postnatal development at P7, P12 or P18. As adults (12 weeks of age), the rats received a second LPS dose (1 mg/kg). Control rats received saline. Microglia were isolated 3 hours post-LPS from the cortex and cervical spinal cord. Gene expression was assessed via qRT-PCR for pro-inflammatory (IL-6, iNOS, Ptgs2, C/EBPb, CD14, CXCL10), anti-inflammatory (CD68, Arg-1), and homeostatic genes (P2Y12, Tmemm119). CSF-1 and CX3CL1 mRNA was analyzed in microglia-free homogenates. Basal gene expression in adult microglia was largely unaffected by early life LPS. Changes in adult microglial pro-inflammatory genes in response to LPS were either unchanged or attenuated in rats exposed to LPS during postnatal development. Ptgs2, C/EBPb, CXCL10 and Arg-1 were the genes most affected, with expression levels significantly downregulated vs control rats without postnatal LPS exposure. Cortical microglia were affected more by postnatal inflammation than spinal microglia, and males were more impacted than females. Overall, inflammatory challenge at P18 had the greatest effect on adult microglial gene expression, whereas challenge at P7 had less impact. Microglial homeostatic genes were unaffected by postnatal LPS. Long-lasting effects of postnatal inflammation on adult microglia depend on the timing of postnatal inflammation, CNS region and sex.

Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection.

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological changes associated with SAP using animal models can aid in identifying potential therapeutic targets and exploring novel treatment approaches. Previous studies primarily induced pancreatic injury through retrograde bile duct injection of sodium taviaurocholate, but the impact of surgical damage on the quality of the animal model remains unclear. In this study, we employed various frequencies of intraperitoneal Caerulein injections combined with different doses of LPS to induce pancreatic injury in C57BL/6J mice and compared the extent of injury across five intraperitoneal injection protocols. Regarding inducing acute pancreatitis in mice, an intraperitoneal injection protocol is proposed that results in a mortality rate as high as 80% within 5 days. Specifically, mice received ten daily intraperitoneal injections of Caerulein (50 µg/kg), followed by an injection of LPS (15 mg/kg) one hour after the last Caerulein administration. By adjusting the frequency and dosage of injected medications, one can manipulate the severity of pancreatic injury effectively. This model exhibits strong controllability and has a short replication cycle, making it feasible for completion by a single researcher without requiring expensive equipment. It conveniently and accurately simulates key disease characteristics observed in human SAP while demonstrating a high degree of reproducibility.

INTERPLAY BETWEEN BRAIN OXYGENATION AND THE DEVELOPMENT OF HYPOTHERMIA IN ENDOTOXIC SHOCK.

There is evidence to suggest that the hypothermia observed in the most severe cases of systemic inflammation or sepsis is a regulated response with potential adaptive value, but the mechanisms involved are poorly understood. Here, we investigated the interplay between brain oxygenation (assessed by tissue P o2 ) and the development of hypothermia in unanesthetized rats challenged with a hypotension-inducing dose of bacterial LPS (1 mg/kg i.v.). At an ambient temperature of 22°C, oxygen consumption (V̇O 2 ) began to fall only a few minutes after the LPS injection, and this suppression in metabolic rate preceded the decrease in core temperature. No reduction in brain P o2 was observed prior to the development of the hypometabolic, hypothermic response, ruling out the possibility that brain hypoxia served as a trigger for hypothermia in this model. Brain P o2 was even increased. Such an improvement in brain oxygenation could reflect either an increased O 2 delivery or a decreased O 2 consumption. The former explanation seems unlikely because blood flow (cardiac output) was being progressively decreased during the recording period. On the other hand, the decrease in V̇O 2 usually preceded the rise in P o2 , and an inverse correlation between V̇O 2 and brain P o2 was consistently observed. These findings do not support the existence of a closed-loop feedback relationship between brain oxygenation and hypothermia in systemic inflammation. The data are consistent with a feedforward mechanism in which hypothermia is triggered (possibly by cryogenic inflammatory mediators) in anticipation of changes in brain oxygenation to prevent the development of tissue hypoxia.

Neutrophil Extracellular Traps Upregulate p21 and Suppress Cell Cycle Progression to Impair Endothelial Regeneration after Inflammatory Lung Injury.

Background: Sepsis is a major cause of ICU admissions, with high mortality and morbidity. The lungs are particularly vulnerable to infection and injury, and restoration of vascular endothelial homeostasis after injury is a crucial determinant of outcome. Neutrophil extracellular trap (NET) release strongly correlates with the severity of lung tissue damage. However, little is known about whether NETs affect endothelial cell (EC) regeneration and repair. Methods: Eight- to ten-week-old male C57BL/6 mice were injected intraperitoneally with a sublethal dose of LPS to induce acute lung inflammatory injury or with PBS as a control. Blood samples and lung tissues were collected to detect NET formation and lung endothelial cell proliferation. Human umbilical vein endothelial cells (HUVECs) were used to determine the role of NETs in cell cycle progression in vitro. Results: Increased NET formation and impaired endothelial cell proliferation were observed in mice with inflammatory lung injury following septic endotoxemia. Degradation of NETs with DNase I attenuated lung inflammation and facilitated endothelial regeneration. Mechanistically, NETs induced p21 upregulation and cell cycle stasis to impair endothelial repair. Conclusions: Our findings suggest that NET formation impairs endothelial regeneration and vascular repair through the induction of p21 and cell cycle arrest during inflammatory lung injury.

Neonatal Cystitis Makes Adult Female Rat Urinary Bladders More Sensitive to Low Concentration Microbial Antigens.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic pain disorder. Patients with IC/BPS often experience "flares" of symptom exacerbation throughout their lifetime, initiated by triggers, such as urinary tract infections. This study sought to determine whether neonatal bladder inflammation (NBI) alters the sensitivity of adult rat bladders to microbial antigens. Female NBI rats received intravesical zymosan treatments on postnatal days P14-P16 while anesthetized; Neonatal Control Treatment (NCT) rats were anesthetized. In adults, bladder and spinal cord Toll-like receptor type 2 and 4 (TLR2, TLR4) contents were determined using ELISAs. Other rats were injected intravesically with lipopolysaccharide (LPS; mimics an E. coli infection; 25, 50, 100, or 200 μg/mL) or Zymosan (mimics yeast infection; 0.01, 0.1, 1, and 10 mg/mL) solutions on the following day. Visceromotor responses (VMRs; abdominal contractions) to graded urinary bladder distention (UBD, 10-60 mm Hg, 20s) were quantified as abdominal electromyograms (EMGs). Bladder TLR2 and TLR4 protein levels increased in NBI rats. These rats displayed statistically significant, dose-dependent, robustly augmented VMRs following all but the lowest doses of LPS and Zymosan tested, when compared with their adult treatment control groups. The NCT groups showed minimal responses to LPS in adults and minimally increased EMG measurements following the highest dose of Zymosan. The microbial antigens LPS and Zymosan augmented nociceptive VMRs to UBD in rats that experienced NBI but had little effect on NCT rats at the doses tested. The greater content of bladder TLR2 and TLR4 proteins in the NBI group was consistent with increased responsiveness to their agonists, Zymosan and LPS, respectively. Given that patients with IC/BPS have a higher incidence of childhood urinary tract infections, this increased responsiveness to microbial antigens may explain the flares in symptoms following "subclinical" tract infections.

Synergy between Iron Deficiency and Inflammation Adversely Affects Pregnancy

Iron is essential for maternal and fetal health during pregnancy. In many developing countries, nutritional deficiencies are common, with anemia affecting upwards of 60% of pregnancies. In addition, infection and inflammation, which are known to be detrimental to pregnancy, are also very prevalent in these regions. Even in high-resource countries, approximately 22% of pregnant women are anemic, and obesity and diabetes are common causes of maternal inflammation. Thus, iron deficiency and inflammation commonly co-exist. In our studies, we noted in mice a striking synergistic adverse interaction between maternal iron deficiency and inflammation that resulted in embryotoxicity, not observed in either condition alone. Considering the high global prevalence of concomitant iron deficiency and inflammation in pregnant women, the issue has outstanding translational importance. We induced iron deficiency in mice by feeding females an iron-poor diet (4ppm) for 1-3 weeks prior to mating and for the duration of pregnancy. This resulted in development of iron deficiency and anemia in both mothers and embryos by term [embryonic day (E)18.5] and placental iron concentrations decreased by half, compared to pregnancies fed control diet (100ppm iron). To model acute systemic maternal inflammation, pregnant females received a single moderate dose of LPS (E. coli serotype O55:B5, 0.5μg/g bodyweight subcutaneously), either in the “2nd” (E8.5) or “3rd” (E15.5) trimester. Embryos and placentas from the E8.5 LPS treatment were collected at E18.5, while those from the E15.5 LPS treatment were collected after 24h. Consistent with other reports, LPS injection alone resulted in some adverse pregnancy outcomes: injection on E8.5 in iron replete dams caused abortion in 22% of dams (total n=9 dams) and at E15.5 caused preterm birth in 50% of dams (n=8) but no embryo malformations or resorption were noted. In contrast, when pregnant dams were both iron-depleted and LPS-injected, we observed a striking synergistic effect resulting in embryo death and resorption in all E8.5 treated pregnancies (n=3 dams). In E15.5-treated pregnancies, 60% of embryos were dead and resorbing (n=42 embryos from 5 dams). This dramatic interaction between iron deficiency and inflammation has not been reported previously. Our data suggest the mechanism appears to be, at least in part, related to activation of the TNFα/TNF-R1 pathway and apoptosis of placental endothelial cells. To test the contribution of the TNFα/TNF-R1 pathway to adverse synergy between iron deficiency and inflammation, we generated endothelial TNF-R1 knockout (KO) mice using two different Cre drivers ( Tnfsfr1 f/f;VEC-Cre + or Tnfsfr1 f/f;Tie2-Cre +). Tnfsfr1 f/f and KO females were iron-depleted and mated with Tnfsfr1 f/fmales. Pregnant dams received a single subcutaneous dose of LPS at E15.5 and pregnancy outcomes were assessed after 24h. Embryos from KO dams were protected from LPS-induced embryotoxicity (30% death, n=7 dams) compared to embryos from WT dams (70% death, n=3 dams). Surprisingly, embryo genotype did not play a role in adverse synergy. Our data suggest that maternal TNFR signaling facilitates adverse interactions between iron deficiency and inflammation during pregnancy, which results in embryo death. These studies are ongoing and our results could provide a mechanistic explanation for known adverse outcomes in complicated human pregnancies, and eventually help inform interventions to optimize outcomes in such pregnancies

Acute Inflammation Redirect Bone Marrow Hematopoietic Stem Cell Cycling and Differentiation Program By Reshaping Their Chromatin Architecture at Long-Term

Background: Acute and persistent inflammation induced by microbial infections/sepsis or by therapeutic interventions, such as whole-body irradiation and CAR-T therapy, triggers a rapid overwhelming immune response in the host. While these responses are well defined in the short-term, the long-term impact of the inflammatory injury on the hematopoietic system still needs to be unveiled. Using a murine model of severe inflammation induced by bacteria P. aeruginosa ( P. aer.) or LPS, our group has previously reported that bone marrow (BM) hematopoietic stem cells (HSC) respond to infections with an aberrant expansion associated with their transitory inability to generate the downstream myeloid progenitors. Such dysfunctions correlated with the suppression of transcription factors governing stem-cell self-renewal and myeloid differentiation. Furthermore, when transplanted into healthy mice, the “inflamed” HSC showed reduced ability to contribute to the myeloid lineage and to engraft at long-term, suggesting that they retain memory of the LPS-mediated insult and maintain long-lasting changes. Thus, we hypothesized that severe inflammation alters the HSC epigenome, reshaping their transcriptional programs and affecting cell fate decisions and differentiation. Methods: We performed time-course experiments in WT mice challenged with a single dose of LPS or PBS, as control, and investigated 1) the kinetics of hematopoietic recovery by flow-cytometric immunophenotypic characterization, including cell-cycle 2) changes in the chromatin landscape of Lin- sorted BM subpopulations identified by the expression of the CD150 and CD48 markers as Long-Term (LT-HSC), Short-Term (ST-HSC), and Multipotent Progenitors (MPP1/2 and MPP3/4) using ATAC-seq. Results: Our studies revealed that the LPS challenge resulted in a significant increase in the cell-cycle of LT-HSC, ST-HSC, MPPs, common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP), which persisted up to 3 days from the inflammatory injury. Immunophenotypic analysis showed that while HSC frequencies returned to baseline at 5 days from LPS, the abundance and cell-cycle of myeloid progenitors and mature cells remained altered. Surprisingly, at 15 days post LPS, the cell-cycle of HSC markedly decreased, and such dysregulation was maintained until our last observation at 30 days from LPS. The pool of BM HSC was severely depleted and associated with exhaustion of T-cells but increased B-cells. GMP and Gr1+Mac1+ cells were also significantly decreased in the BM whereas mature lineages were increased in the peripheral blood, exhibiting high WBC, lymphocytes, and platelet count, neutrophilia, monocytosis, basophilia, and eosinophilia. ATAC-seq analysis revealed prominent differences in the chromatin architecture of the four HSC subpopulations at steady state (PBS). In all of them, LPS induced both i) a transient reorganization of the chromatin in some loci, and ii) the formation of persistently reshaped regions. Deeper investigations into the long-term changed sites revealed that chromatic accessibility was altered in proximity of CTCF-enriched regions fundamental to mediate chromatin reorganization and drive gene expression. Moreover, LPS modified the accessibility of genomic loci involved in the maintenance of HSC stemness and quiescence as well as regulation of their cell-cycle, self-renewal, and cell fate decision. Conclusions: Taken together these results suggest that LPS-mediated acute inflammation has long-lasting effects on HSC epigenetic regulation, influencing their cell-cycle and multilineage decisions. Relevance: The association between the resulting phenotype and the persistency of epigenetically remodeled HSC loci identified here may provide potential insights for future therapeutic approaches to re-direct “inflamed” HSC to a normal differentiation program, thereby enabling an efficient innate host response. Disclosures: No relevant conflicts of interest to declare.

Sepsis induced cardiotoxicity by promoting cardiomyocyte cuproptosis

BackgroundCurrently, sepsis induced cardiotoxicity is among the major causes of sepsis-related death. The specific molecular mechanisms of sepsis induced cardiotoxicity are currently unknown. Therefore, the purpose of this paper is to identify the key molecule mechanisms for sepsis induced cardiotoxicity. MethodsOriginal data of sepsis induced cardiotoxicity was derived from Gene Expression Omnibus (GEO; GSE63920; GSE44363; GSE159309) dataset. Functional enrichment analysis was used to analysis sepsis induced cardiotoxicity related signaling pathways. Our findings also have explored the relationship of cuproptosis and N6-Methyladenosine (m6A) in sepsis induced cardiotoxicity.Mice are randomly assigned to 3 groups: saline treatment control group, LPS group administered a single 5 mg/kg dose of LPS for 24 h, LPS + CD274 inhibitor group administered 10 mg/kg CD274 inhibitor for 24 h. ResultsOverall, expression of cuproptosis-related genes (CRGs) CD274, Ceruloplasmin (CP), Vascular endothelial growth factor A (VEGFA), Copper chaperone for cytochrome c oxidase 11 (COX11), chemokine C–C motif ligand 8 (CCL8), Mitogen-activated protein kinase kinase 1(MAP2K1), Amine oxidase 3 (AOC3) were significantly altered in sepsis induced cardiotoxicity. The results of spearman correlation analysis was significant relationship between differentially regulated genes (DEGs) of CRGs and the expression level of m6A methylation genes. GO and KEGG showed that these genes were enriched in response to interferon-beta, MHC class I peptide loading complex, proteasome core complex, chemokine receptor binding, TAP binding, chemokine activity, cytokine activity and many more. These findings suggest that cuproptosis is strongly associated with sepsis induced cardiotoxicity. ConclusionIn the present study, we found that cuproptosis were associated with sepsis induced cardiotoxicity. The CD274, CP, VEGFA, COX11, CCL8, MAP2K1, AOC3 genes are showing a significant difference expression in sepsis induced cardiotoxicity. Our studies have found significant correlations between CRGs and m6A methylation related genes in sepsis induced cardiotoxicity. These results provide insight into mechanism for sepsis induced cardiotoxicity.

Progression of Acute Lung Injury in Intratracheal LPS Rat Model: Efficacy of Fluticasone, Dexamethasone, and Pirfenidone

Introduction: We investigated the potential of LPS (10–300 µg/rat) administered intratracheally (i.t.) to induce reproducible features of acute lung injury (ALI) and compared the pharmacological efficacy of anti-inflammatory glucocorticoids and antifibrotic drugs to reduce the disease. Additionally, we studied the time-dependent progression of ALI in this LPS rat model. Methods: We conducted (1) dose effect studies of LPS administered i.t. at 10, 30, 100, and 300 μg/rat on ALI at 4 h timepoint; (2) pharmacological interventions using i.t. fluticasone (100 and 300 μg/rat), i.t. pirfenidone (4,000 μg/rat), and peroral dexamethasone (1 mg/kg) at 4 h timepoint; (3) kinetic studies at 0, 2, 4, 6, 8, 10, and 24 h post-LPS challenge. Phenotype or pharmacological efficacy was assessed using predetermined ALI features such as pulmonary inflammation, edema, and inflammatory mediators. Results: All LPS doses induced a similar increase of inflammation, edema, and inflammatory mediators, e.g., IL6, IL1β, TNFα, and CINC-1. In pharmacological intervention studies, we showed fluticasone and dexamethasone ameliorated ALI by inhibiting inflammation (>60–80%), edema (>70–100%), and the increase of cytokines IL6, IL1β, and TNFα (≥70–90%). We also noticed some inhibition of CINC-1 (25–35%) and TIMP1 (57%) increase with fluticasone and dexamethasone. Conversely, pirfenidone failed to inhibit inflammation, edema, and mediators of inflammation. Last, in ALI kinetic studies, we observed progressive pulmonary inflammation and TIMP1 levels, which peaked at 6 h and remained elevated up to 24 h. Progressive pulmonary edema started between 2 and 4 h and was sustained at later timepoints. On average, levels of IL6 (peak at 6–8 h), IL1β (peak at 2–10 h), TNFα (peak at 2 h), CINC-1 (peak at 2–6 h), and TGFβ1 (peak at 8 h) were elevated between 2 and 10 h and declined toward 24 h post-LPS challenge. Conclusion: Our data show that 10 μg/rat LPS achieved a robust, profound, and reproducible experimental ALI phenotype. Glucocorticoids ameliorated key ALI features at the 4-h timepoint, but the antifibrotic pirfenidone failed. Progressive inflammation and sustained pulmonary edema were present up to 24 h, whereas levels of inflammatory mediators were dynamic during ALI progression. This study’s data might be helpful in designing appropriate experiments to test the potential of new therapeutics to cure ALI.

Abstract 317: Development of a Swine Model of Rapidly-Induced Septic Shock Using Single Lipopolysaccharide Infusion and Titratable Inhaled Nitric Oxide to Prevent Severe Pulmonary Vasoconstriction

Objective: A serious limitation of LPS-induced swine models of septic shock is severe pulmonary vasoconstriction triggered, in part, by the pulmonary release of thromboxane A2. Efforts to overcome this limitation have included a stepwise increase in LPS dosing, intravenous aspirin derivatives, and inhaled nitric oxide (NO). We studied the effects of titrated inhaled NO given coincident with rapid LPS-infusion aimed at inducing the key features of septic shock over a shorter time period. Methods: We developed a LabVIEW-controlled system for inhaled NO delivery and monitoring. Exploratory studies showed that an NO concentration of up to 300 ppm sufficed to prevent significant pulmonary vasoconstriction despite delivery of a full LPS dose (12.6 μg/kg) in 30 minutes. We used this approach in 6 male domestic pigs (39.2 - 41.1 kg) and assessed hemodynamic and metabolic effects over 6 hours. NO and LPS infusion were started concurrently. Fluid resuscitation and vasopressor therapy were guided by macrohemodynamic measurements using custom-developed LabVIEW-controlled closed-loop algorithms. Results: Shown are medians with IQ25 and IQ75. LPS infusion increased pulmonary vascular resistance from 112 [72, 127] at baseline to 213 [120, 313] dynes-sec/cm 5 after 20 minutes. NO (139 [131, 166] ml/min) rapidly blunted the effect preventing the mean pulmonary artery pressure from exceeding 30 mmHg, avoiding detrimental effects on right ventricular function throughout the experiment. NO was rapidly de-escalated over 30 minutes using a closed-loop algorithm developed to prevent the mean pulmonary artery pressure from exceeding 30 mmHg. Within 60 minutes of LPS infusion start, animals began to develop key features of septic shock including tachycardia, hypotension, and systemic vasodilation triggering fluid resuscitation and norepinephrine administration. Additionally, blood lactate increased from 1.06 [0.90, 1.45] at baseline to 4.14 [2.43, 8.78] mmol/l after 3 hours remaining stable for an additional hour and subsequently decreasing in 3 and increasing in the other 3 animals, with half of the animals surviving the 6-h period. Conclusion: We developed a swine model of rapidly-induced key features of septic shock useful to explore early septic shock management.

PSIV-4 Impact of Lipopolysaccharide Endotoxin on Growth and Viability of Ovarian Granulosa Cells

Abstract Lipopolysaccharide (LPS) endotoxin from Gram-negative bacteria has been demonstrated to negatively impact reproductive parameters leading to ovarian dysfunction. Ovarian granulosa cells express the toll-like receptor 4 complex necessary to elicit inflammatory immune responses in the presence of LPS. Quantifiable amounts of LPS have been detected in follicular fluid from slaughterhouse-derived ovaries with further deleterious effects on granulosa cell steroidogenesis. Therefore, the objective of this study was to investigate the effects of LPS on granulosa cell proliferation in vitro. We hypothesized that increasing concentrations of LPS would negatively impact granulosa cell proliferation dynamics. A human granulosa cell line (KGN) was plated at 60% confluency in 96-well tissue culture plates and treated with phosphate buffered saline-vehicle or LPS (0.01, 0.1, 1, or 10 µg/mL) in the presence or absence of follicle-stimulating hormone (FSH). Cells were incubated for 48 h at 37 ºC and 5% CO2 in an IncuCyte incubator that utilizes real-time automated microscopy to continuously capture images of cells and measure cell confluency. Individual well images were collected every 6 h. The study was performed in 3 independent experiments with 8 replicates per treatment group in each experiment. Cells were allowed to equilibrate in the IncuCyte for 6 h before image collection. Cell confluency for each treatment group was normalized to vehicle-treated control confluency at 6 h. A significant difference was observed at the initial 6 h timepoint, with 1 and 10 µg/mL LPS treatment groups demonstrating decreased cell confluency compared with vehicle-treated controls (P &amp;lt; 0.05). However, no significant difference was observed in 0.01 (P = 0.65) and 0.1 (P = 0.53) µg/mL LPS treatment groups compared with vehicle-treated controls at 6 h timepoint. Interestingly, the deleterious effects of LPS on confluency and cell growth parameters were mitigated with the addition of FSH. No observable differences were detected in the two largest LPS doses concurrently incubated with FSH, 1 and 10 µg/mL LPS (P = 0.55 and P = 0.62, respectively), compared with vehicle-treated controls. From 12 h until the end of the incubation period, cell confluency in 1 and 10 µg/mL LPS treatment groups remained significantly different compared with FSH-treated cells (P &amp;lt; 0.05). Comparably, no differences were detected among FSH-stimulated groups, regardless of LPS dose or time (P = 0.99). Results indicate that greater LPS concentrations (1 and 10 µg/mL) negatively impacts cell confluency and proliferation in vitro; however, FSH has the ability to protect granulosa cells against the detrimental effects that LPS has on cell confluency. These data suggest that FSH is critical in regulating cell growth and proliferation despite the action of inflammatory pathways induced by bacterial endotoxin.

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.

Isomaculosidine facilitates NLRP3 inflammasome activation by promoting mitochondrial reactive oxygen species production and causes idiosyncratic liver injury

Ethnopharmacological relevanceDictamnus dasycarpus Turcz. (Dictamni Cortex, DC), a Chinese herbal medicine, is commonly used for treating chronic dermatosis and rheumatism, but can also cause herb-induced liver injury (HILI). Our study has demonstrated that DC can induce idiosyncratic HILI, but the mechanism remains unknown. The NLRP3 inflammasome has become a major target for addressing many diseases. The activation of NLRP3 inflammasome is responsible for many liver-related inflammatory diseases, including idiosyncratic HILI. Aim of the studyThe objective of our study was to demonstrate the mechanism underlying the idiosyncratic HILI induced by DC and clarify the susceptible component in DC. Materials and methodsBone marrow-derived macrophages (BMDMs) and THP1 cells were selected to assess the effect of isomaculosidine (IMD) on NLRP3 inflammasome activation in vitro. Western blot, ELISA and Caspase-Glo® 1 Inflammasome Assay, flow cytometry and Immunofluorescence were employed to detect the mechanism of IMD on NLRP3 inflammasome activation. To assess the efficacy of IMD in vivo, mice were intravenously administrated with LPS and then IMD were injected intraperitoneally for 6 h. ResultsThe results of our in vitro studies demonstrate that IMD, the major constituent of DC, specifically promoted ATP- and nigericin-induced activation of NLRP3 inflammasome, but not NLRC4 and AIM2 inflammasomes. Additionally, IMD promoted nigericin-induced ASC oligomerization. Notably, synergistic induction of mtROS played a key role on the activation of NLRP3 inflammasome. IMD increased the mtROS production in the activation of NLRP3 inflammasome induced by nigericin. In addition, the results of our in vivo study showed that the combination of nonhepatotoxic doses of LPS and IMD can increase the levels of ALT, AST, and DBIL, leading to liver injury. ConclusionsIMD specifically facilitated the activation of NLRP3 inflammasome induced by nigericin and ATP, which is responsible for DC-induced idiosyncratic HILI.