Chronic Lipopolysaccharide Research Articles

8698 Unique populations of pituitary macrophages regulate gonadotropin secretion

Abstract Disclosure: Z. Del Mundo: None. J. Ha: None. A. Zhang: None. K.D. Wiggins: None. G. De Robles: None. C. Garcia: None. N. Ujagar: None. D. Nicholas: None. Chronic inflammation disrupts hormonal balance in the Hypothalamic-Pituitary-Gonadal (HPG) axis, contributing to reproductive disorders. Despite extensive studies on immune cells in the hypothalamus and ovaries, their impact on the pituitary is largely unexplored. Our research reveals that macrophages are the primary immune population in the pituitary during homeostasis. In response to in vivo chronic lipopolysaccharide exposure in mice, a transient macrophage population emerges, associated with reproductive changes, including elevated serum testosterone, prolonged estrous cycle stage, and reduced ovarian antral follicle count. This study aims to characterize pituitary macrophages and elucidate their impact on the HPG axis. We hypothesize that pituitary macrophages change inflammatory status as a response to stimuli and, as a result, modulate gonadotropin secretion. We pinpointed differentially expressed genes (DEGs) in pituitary macrophages using scRNAseq, distinguishing them from other tissue-resident macrophages, including microglia. These DEGs include markers for macrophage activation, phagocytosis, migration, hormone synthesis, and lipid metabolism. Immunohistochemistry validated the distinctive morphology of pituitary macrophages compared to microglia. In vitro depletion of macrophages from mouse pituitaries resulted in decreased pituitary hormone secretion and decreased secretion of G-CSF, IL-9, and CXCL5. These results highlight the role of pituitary macrophages in hormonal regulation, setting the stage for future immunotherapeutic strategies to restore hormonal balance in reproductive disorders. Presentation: 6/3/2024

Chronic inflammation decreases arcuate kisspeptin expression in male sheep

Lipopolysaccharide (LPS) from Gram-negative bacteria induces an immune response and impairs reproduction through suppression of gonadotropin releasing hormone (GnRH), subsequently luteinizing hormone (LH) secretion. While there is evidence that acute inflammation inhibits kisspeptin, little is known about the impact of chronic inflammation on this key reproductive neuropeptide in livestock species. Thus, we sought to examine a central mechanism whereby LPS suppresses LH secretion in sheep. Twenty wethers were randomly assigned to one of five treatment groups: control (CON; n=4), single acute IV LPS dose (SAD; n=4), daily acute IV LPS dose (DAD; n=4), daily increasing IV LPS dose (DID; n=4), and chronic subcutaneous LPS dose (CSD; n=4). On Days 1 and 7, blood samples were collected every 12 minutes for 360 minutes using jugular venipuncture. Following blood collection on Day 7, all animals were euthanized, brain tissue was perfused with 4% paraformaldehyde, and hypothalamic blocks were removed and processed for immunohistochemistry. On Day 1, LH pulse frequency was significantly lower (p=0.02) in SAD (0.25 ± 0.1 pulses/hour), DAD (0.25 ± 0.1 pulses/hour), DID (0.35 ± 0.1 pulses/hour), and CSD (0.40 ± 0.1 pulses/hour) compared to CON (0.70 ±0.1 pulses/hour). On Day 7, only DID animals (0.35 ± 0.1 pulses/hour) had significantly lower (p=0.049) LH pulse frequency compared to controls (0.85 ± 0.1 pulse/hour). Furthermore, only DID animals (33.3 ± 10.9 cells/section/animal) had significantly fewer (p=0.001) kisspeptin-immunopositive cells compared to controls (82.6 ± 13.6 cells/section/animal). Taken together, we suggest that daily increasing doses of LPS is a powerful inhibitor of kisspeptin neurons in young male sheep and a physiologically relevant model to examine the impact of chronic inflammation on the reproductive axis in livestock.

Comparison of chronic restraint stress-and lipopolysaccharide-induced mouse models of depression: Behavior, c-Fos expression, and microglial and astrocytic activation

Depression is a mental disease that involves a variety of complex physiological mechanisms. A wide range of methods have therefore been used to establish mouse models of depression, and there are currently many ways to develop such mouse models. The present study aimed to compare the effects of various model induction methods and assesses their different effects. To this end, C57BL/6J mice were divided into three experimental groups: the chronic restraint stress (CRS) group received 6 hours of daily confinement within restraint tubes over a 3-week period; the chronic lipopolysaccharide (C-LPS) administration group received daily intraperitoneal injections of 0.5 mg/kg LPS for 1 week; and the acute LPS (A-LPS) administration group received a singular intraperitoneal injection of 0.83 mg/kg LPS. A corresponding control group was established for each experimental condition. Following mouse model establishment, depression-like behaviors were assessed through the forced swimming and tail suspension tests; anxiety-related behaviors were evaluated using the open field test and elevated plus maze. Furthermore, the expression of the immediate early gene c-Fos, ionized calcium-binding adapter molecule 1 (IBA1), and glial fibrillary acidic protein (GFAP) was examined via immunofluorescence. Longer immobility durations during the forced swimming and tail suspension tests were observed across all model groups (p < 0.05), indicating depression-like behaviors. Furthermore, the CRS and C-LPS group, but not the A-LPS group, showed significant anxiety-like behaviors in the elevated plus maze (p < 0.05). All model groups also exhibited significant increases in both time and distance explored within the central area of the open field test (p < 0.05). The activation of GFAP- and IBA1-positive cells in the cerebral cortex and hippocampus was also markedly pronounced in all experimental groups, suggesting the association of neuroinflammatory responses with induced depressive states. The present findings contribute to our understanding of the pathophysiology of stress-induced and neuroinflammatory-associated depression, and will help researchers to choose suitable depression models for their investigations.

Possible Prophylactic Effects of Sulforaphane on LPS-Induced Recognition Memory Impairment Mediated by Regulating Oxidative Stress and Neuroinflammatory Proteins in the Prefrontal Cortex Region of the Brain.

Alzheimer's disease (AD) presents a significant global health concern, characterized by neurodegeneration and cognitive decline. Neuroinflammation is a crucial factor in AD development and progression, yet effective pharmacotherapy remains elusive. Sulforaphane (SFN), derived from cruciferous vegetables and mainly from broccoli, has shown a promising effect via in vitro and in vivo studies as a potential treatment for AD. This study aims to investigate the possible prophylactic mechanisms of SFN against prefrontal cortex (PFC)-related recognition memory impairment induced by lipopolysaccharide (LPS) administration. Thirty-six Swiss (SWR/J) mice weighing 18-25 g were divided into three groups (n = 12 per group): a control group (vehicle), an LPS group (0.75 mg/kg of LPS), and an LPS + SFN group (25 mg/kg of SFN). The total duration of the study was 3 weeks, during which mice underwent treatments for the initial 2 weeks, with daily monitoring of body weight and temperature. Behavioral assessments via novel object recognition (NOR) and temporal order recognition (TOR) tasks were conducted in the final week of the study. Inflammatory markers (IL-6 and TNF), antioxidant enzymes (SOD, GSH, and CAT), and pro-oxidant (MDA) level, in addition to acetylcholine esterase (AChE) activity and active (caspase-3) and phosphorylated (AMPK) levels, were evaluated. Further, PFC neuronal degeneration, Aβ content, and microglial activation were also examined using H&E, Congo red staining, and Iba1 immunohistochemistry, respectively. SFN pretreatment significantly improved recognition memory performance during the NOR and TOR tests. Moreover, SFN was protected from neuroinflammation and oxidative stress as well as neurodegeneration, Aβ accumulation, and microglial hyperactivity. The obtained results suggested that SFN has a potential protective property to mitigate the behavioral and biochemical impairments induced by chronic LPS administration and suggested to be via an AMPK/caspase-3-dependent manner.

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

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

Disrupted Insulin Signaling Due to an Acute Immune Response in Swine

Insulin is an anabolic hormone involved in glucose uptake and synthesis of fats, proteins, and glycogen. Altered insulin signaling can occur due to metabolic state, pathogen exposure, and autoimmune disorders. Lipopolysaccharide (LPS), an endotoxin secreted produced by gram-negative bacteria, can induce a severe, systemic immune response. In pigs, chronic LPS exposure has induced insulin resistance. However, the effects of acute exposure to LPS on insulin signaling and resistance has not been elucidated. Crossbred nursery pigs (58, 12.1 kg ± 0.23kg BW) were randomly assigned into two experimental subsets, where 10 animals were designated for repeated blood sampling and 48 animals underwent timed euthanasia for tissue collection. For repeated blood sampling, jugular catheters were placed under anesthesia and the animals were given 48 hours to recover before being randomly assigned to treatments. These treatments were an intraperitoneal injection of either 15μg/kg BW LPS or an equal volume of 0.9% Saline (n=5 pigs/treatment). Blood was collected for 48 hours followed by euthanasia. The other 48 pigs were randomly assigned to the same treatments, and then randomly assigned to euthanasia timepoints of 0, 3, 6, 12, 24 or 48 hours post injection for tissue collection. Among the catheterized pigs, two hours post LPS challenge there was a decrease in serum insulin concentration (p&lt;0.05) and mild hyperglycemia (p&lt;0.05) post challenge. At 12 and 24 hours post LPS challenge there was a decrease in insulin (p&lt;0.05) and a trend hyperglycemia was also apparent at 24 hours (p=0.063). In liver tissue, insulin related genes exhibited alterations post LPS injection. Phosphoinositide 3-kinase ( pi3k) and insulin receptor substrate 1 ( irs1) expression were decreased at 3 and 6 hours (p&lt;0.05) and protein kinase b ( akt) expression were decreased at 3 hours (p&lt;0.05) post challenge. The expression of insulin inhibitor growth factor receptor bound protein 10 ( grb10) was decreased at 6, 12, and 24 hours after challenge (p&lt;0.05). Fructose-1-6-bisphosphatase ( f16bp) expression was reduced at 3, 6, and 12 hours (p&lt;0.05) in the liver while phosphoenolpyruvate carboxykinase ( pepck) expression was decreased at 3 and 6 hours (p&lt;0.01) post challenge. Other metabolic genes were decreased at 3, 6, and 12 hours post LPS challenge, including fatty acid synthase ( fas) (p&lt;0.05), carbohydrate response element binding protein ( chrebp) (p&lt;0.05), and acetyl CoA carboxylase ( acc)(p&lt;0.05). Protein abundance of Akt 3 hours after LPS injection was decreased in the liver (p&lt;0.05). In skeletal muscle 6 hours post LPS injection, insulin receptor ( insr), insulin-like growth factor receptor ( igfr) and fas were all increased (p&lt;0.05) These results indicate disrupted insulin signaling at various levels after an acute immune response. Research reported in this abstract was supported by Eunice Kennedy Shriver National Institute of Child Health &amp; Human Development (NICHD) of the National Institutes of Health under award number R01HD092304A. 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 alleviates chronic inflammation-induced neuronal ferroptosis and cognitive impairments via regulation of AIM2 - Nrf2 signaling pathway

Ethnopharmacological relevanceGinseng is a valuable herb in traditional Chinese medicine. Modern research has shown that it has various benefits, including tonifying vital energy, nourishing and strengthening the body, calming the mind, improving cognitive function, regulating fluids, and returning blood pressure, etc. Rg1 is a primary active component of ginseng. It protects hippocampal neurons, improves synaptic plasticity, enhances cognitive function, and boosts immunity. Furthermore, it exhibits anti-aging and anti-fatigue properties and holds great potential for preventing and managing neurodegenerative diseases (NDDs). Aim of the studyThe objective of this study was to examine the role of Rg1 in treating chronic inflammatory NDDs and its molecular mechanisms. Materials and methodsIn vivo, we investigated the protective effects of Rg1 against chronic neuroinflammation and cognitive deficits in mice induced by 200 μg/kg lipopolysaccharide (LPS) for 21 days using behavioral tests, pathological sections, Western blot, qPCR and immunostaining. In vitro experiments involved the stimulation of HT22 cells with 10 μg/ml of LPS, verification of the therapeutic effect of Rg1, and elucidation of its potential mechanism of action using H2DCFDA staining, BODIPY™ 581/591 C11, JC-1 staining, Western blot, and immunostaining. ResultsFirstly, it was found that Rg1 significantly improved chronic LPS-induced behavioral and cognitive dysfunction in mice. Further studies showed that Rg1 significantly attenuated LPS-induced neuronal damage by reducing levels of IL-6, IL-1β and ROS, and inhibiting AIM2 inflammasome. Furthermore, chronic LPS exposure induced the onset of neuronal ferroptosis by increasing the lipid peroxidation product MDA and regulating the ferroptosis-associated proteins Gpx4, xCT, FSP1, DMT1 and TfR, which were reversed by Rg1 treatment. Additionally, Rg1 was found to activate Nrf2 and its downstream antioxidant enzymes, such as HO1 and NQO1, both in vivo and in vitro. In vitro studies also showed that the Nrf2 inhibitor ML385 could inhibit the anti-inflammatory, antioxidant, and anti-ferroptosis effects of Rg1. ConclusionsThis study demonstrated that Rg1 administration ameliorated chronic LPS-induced cognitive deficits and neuronal ferroptosis in mice by inhibiting neuroinflammation and oxidative stress. The underlying mechanisms may be related to the inhibition of AIM2 inflammasome and activation of Nrf2 signaling. These findings provide valuable insights into the treatment of chronic neuroinflammation and associated NDDs.

Serum Lipopolysaccharide-binding Protein Levels and the Incidence of Metabolic Syndrome in a General Japanese Population: the Hisayama Study.

The association between chronic lipopolysaccharide exposure and the development of metabolic syndrome (MetS) is unclear. In this study we examined the association between serum lipopolysaccharide-binding protein (LBP) levels, an indicator of lipopolysaccharide exposure, and the development of MetS in a general Japanese population. 1,869 community-dwelling Japanese individuals aged ≥40 years without MetS at baseline examination in 2002-2003 were followed up by repeated examination in 2007-2008. MetS was defined according to the Japanese criteria. Serum LBP levels were classified into quartiles (quartiles 1-4: 2.20-9.56, 9.57-10.78, 10.79-12.18, and 12.19-24.34 µg/mL, respectively). Odds ratios (ORs) for developing MetS were calculated using a logistic regression model. At the follow-up survey, 159 participants had developed MetS. Higher serum LBP levels were associated with greater risk of developing MetS after multivariable adjustment for age, sex, smoking, drinking, and exercise habits (OR [95% confidence interval] for quartiles 1-4: 1.00 [reference], 2.92 [1.59-5.37], 3.48 [1.91-6.35], and 3.86 [2.12-7.03], respectively; P for trend <0.001). After additional adjustment for homeostasis model assessment of insulin resistance, this association was attenuated but remained significant (P for trend = 0.007). On the other hand, no significant association was observed after additional adjustment for serum high-sensitivity C-reactive protein (P for trend = 0.07). In the general Japanese population, our findings suggest that higher serum LBP levels are associated with elevated risk of developing MetS. Low-grade endotoxemia could play a role in the development of MetS through systemic chronic inflammation and insulin resistance.

Mitigating Oxidative Stress Promotes Quiescence of Hematopoietic Stem Cells from Concurrent TLR4 Activation and IL-10R Blockade Mediated Inflammation

Inflammatory stress provides a clonogenic advantage towards CHIP mutant hematopoietic stem and progenitor cells (HSPCs) via different mechanisms including resistance to apoptosis, enhanced self-renewal and limited generation of reactive oxygen species. Reactive oxygen species (ROS) play a causative role in hematopoietic stem cell (HSC) dysfunction due to exit from quiescence and replication stress-induced stem cell exhaustion. Oxidative stress mediated decline in the fitness of WT hematopoietic stem cells (HSCs) may allow mutant HSCs to gain a selective advantage. Therefore, protection of WT HSCs from ROS may maintain their fitness, and prevent the selective expansion of mutant cells. To test whether the anti-oxidant N-Acetylcysteine (N-AC) protects HSCs from chronic inflammatory stress, we mice were challenged daily with lipopolysaccharide (LPS) (5μg, intraperitoneal) or saline control for 30 days while their drinking water was supplemented with N-AC at 2mg/ml or untreated regular water. At day 30, bone marrow was harvested for competitive repopulation assays using equal numbers of whole bone marrow cells from each competitor (Figure 1A). As expected, bone marrow from mice exposed to 30 days of chronic LPS on regular water showed inferior reconstitution compared to saline exposed mice. However, bone marrow from mice exposed to 30 days of LPS and maintained on N-AC supplemented water performed equivalently in competitive transplants as saline exposed mice. To demonstrate that this functional defect of HSCs stemming from chronic LPS treatment is dependent on increased HSC divisions, we challenged TetOP-H2B-GFP mice with LPS for 30 days and assessed H2B-GFP label retention in immunophenotypically defined long-term HSCs. Progenitor populations in the bone marrow demonstrated low levels of H2B-GFP expression while LT-HSCs showed high GFP signal indicating low turnover. Meanwhile, LPS treatment led to increased cell divisions in LT-HSCs compared to saline treated mice. We hypothesized that N-AC protects HSCs via reducing entry into cell cycle in response to inflammatory stimuli, which is ROS dependent. We assessed the impact of N-AC treatment on ROS in HSCs and its correlation with cell cycling. To induce acute inflammation, wildtype (WT) mice were injected with LPS (5μg) and IL-10R blocking antibody (0.1mg). IL-10R blocking antibody was used in conjunction with LPS because we have previously found that IL-10R blockade extends LPS induced HSC proliferation. Intracellular ROS was determined using the ROS sensitive dye, CM-H 2DCFDA (DCF). The DCF signal was increased in HSCs (Lin -, c-Kit +, Sca1 +, CD150 +, CD48 -) in response to TLR4 activation by LPS and the addition of αIL-10R further augmented this increase in ROS (Figure 1B). Pre-treatment with N-AC significantly reduced the increase in ROS mediated by LPS alone. Cell cycle status was determined by intracellular Ki67 staining of HSCs. LPS significantly increased HSC proliferation as determined by increased percentage of cells in the G1 phase of the cell cycle while IL-10R blockade further augmented HSC cycling. Mice pre-treated with N-AC did not show significant increase in HSC proliferation indicating protection from ROS mediated exit from quiescence. In conclusion, incorporation of N-AC into drinking water protected WT HSCs from inflammation induced exhaustion and promoted persistence of quiescence in HSCs. N-AC could potentially be utilized as a preventative method to preserve fitness of HSCs, particularly in those with chronic inflammatory stress. Moreover, preservation of HSC fitness should be evaluated for its ability to prevent emergence of clonal hematopoiesis.

SAT385 Pituitary Immune Cells Regulate Gonadotrope Hormone Secretion

Abstract Disclosure: Z. Del Mundo: None. T. Nguyen: None. N. Ujagar: None. D. Nicholas: None. Polycystic ovary syndrome (PCOS) is one of the most common causes of female infertility in the US and is associated with a combination of reproductive neuroendocrine physiological changes. Previous studies have shown that PCOS patients have elevated serum C reactive peptide and lipopolysaccharide (LPS), which indicates chronic low-grade inflammation. However, it is unknown how inflammation or immune cells impact reproductive outcomes. In this project, I study how inflammation affects gonadotrope regulation of sex hormone secretion. I show that in vitro depletion of immune cells from dispersed primary mouse pituitary increases the luteinizing hormone (LH) to follicle-stimulating hormone (FSH) ratio. In contrast, immune activation through chronic LPS exposure in mice results in decreased serum LH to FSH ratio. Using single-cell RNA sequencing data from immune cell-enriched mouse pituitaries, I identified specific resident immune cell populations. These cell populations include macrophages with higher RNA expression of heat shock proteins in male mice compared to female mice suggesting inflammatory protection in males. Ongoing experiments include chronic LPS exposure in mice, where pituitary cells are immunophenotyped, sex hormone serum levels measured, and ovulation monitored. Ultimately this work will allow us to understand the contribution of inflammation to PCOS, with the hope of designing immunotherapy for PCOS. Presentation Date: Saturday, June 17, 2023

DNMT3B Alleviates Liver Steatosis Induced by Chronic Low-grade LPS via Inhibiting CIDEA Expression

Nonalcoholic fatty liver disease is the most prevalent chronic liver disease and threats to human health. Gut dysbiosis caused by lipopolysaccharide (LPS) leakage has been strongly related to nonalcoholic fatty liver disease progression, although the underlying mechanisms remain unclear. Previous studies have shown that low-grade LPS administration to mice on a standard, low-fat chow diet is sufficient to induce symptoms of fatty liver. This study confirmed these findings and supported LPS as a lipid metabolism regulator in the liver. Mechanically, LPS induced dysregulated lipid metabolism by inhibiting the expression of DNA methyltransferases 3B (DNMT3B). Genetic overexpression of DNMT3B alleviated LPS-induced lipid accumulation, whereas its knockdown increased steatosis in mice and human hepatocytes. LPS-induced lower expression of DNMT3B led to hypomethylation in promoter region of CIDEA, resulting in increased binding of SREBP-1c to its promoter and activated CIDEA expression. Hepatic interference of CIDEA reversed the effect of LPS on lipogenesis. These effects were independent of a high-fat diet or high fatty acid action. Overall, these findings sustain the conclusion that LPS is a lipogenic factor and could be involved in hepatic steatosis progression.

Pituitary immune cells regulate gonadotrope hormone secretion

Abstract Polycystic ovary syndrome (PCOS) is one of the most common causes of female infertility in the US and is associated with a combination of reproductive neuroendocrine physiological changes. Previous studies have shown that PCOS patients have elevated serum C reactive peptide and lipopolysaccharide (LPS), which indicates chronic low-grade inflammation. However, it is unknown how inflammation or immune cells impact reproductive outcomes. In this project, I study how inflammation affects gonadotrope regulation of sex hormone secretion. I show that in vitro depletion of CD45+ cells from dispersed primary mouse pituitary increases the luteinizing hormone (LH) to follicle-stimulating hormone (FSH) ratio. In contrast, immune activation through chronic LPS exposure in mice results in decreased serum LH to FSH ratio. Using single-cell RNA sequencing data from CD45+ enriched cells from mouse pituitaries, I identified specific resident immune cells. These cell populations include macrophages with higher RNA expression of heat shock proteins in male mice compared to female mice suggesting inflammatory protection in males. Ongoing experiments include chronic LPS exposure in mice, where pituitary cells are immunophenotyped, sex hormone serum levels measured, and ovulation monitored. Ultimately this work will allow us to understand the contribution of inflammation to PCOS, with the hope of designing immunotherapy for PCOS. NICHD R00 HD098330-03

Acute and chronic lipopolysaccharide-induced stress changes expression of proinflammatory cytokine genes in the rat brain region-specifically and affects learning and memory

The purpose of the current work was a comparative analysis of the effects of acute and chronic lipopolysaccharide stress on behavior of rats in the Morris water maze and expression of mRNA proinflammatory cytokines and BDNF in different brain structures. The relevance of this study is related with a weak knowledge of the effects of acute and chronic stress on manifestation of cognitive brain functions, ambiguity of influences of both stresses on the hypothalamic-pituatary axis and expression of the proinflammatory cytokines genes, as well as contradictory data in the literature. In experiments on rats, the acute lipopolysaccharide (LPS) stress improved learning in the Morris water maze. For the period of learning, the rats swam on average less distance to reach a hidden platform and spent less time in the outer zone of the pool (tigmotaxis) and had a low speed compared to the control animals and a group of rats with chronic LPS stress. In a probe trial without a platform in the pool there were no significant differences between groups on time spent in the platform quadrant and distance swum. The acute stress produced a substantial increase of TNF-α and IL-1β concentration in the hippocampus and amygdala, but not in the frontal cortex relative to the control animals. Although the chronic stress increased the levels of TNF-α and IL-1β in the amygdala and hippocampus compared to the control groups, the significance between the groups was only marginal and the concentration of BDNF did not differ from the control animals in none of the structures mentioned. The concentration of IL-6 marginally increased in acute LPS stress in the amygdala and marginally decreased chronic LPS stress in the hippocamus relative to the saline control groups. In total, the most clear molecular-biochemical changes occurred in the amygdala and hippocampus, where the increase of interleukines TNF-α and IL-1β were seen in the acute and chronic LPS stress and no changes in BDNF concentration in the frontal cortex.

Acute and Chronic Lipopolysaccharide-Induced Stress Changes Expression of Proinflammatory Cytokine Genes in the Rat Brain Region-Specifically and Affects Learning and Memory

Goal of the current work was to conduct comparative analysis of the effects of acute and chronic lipopolysaccharide-induced stress on the behavior of rats in the Morris water maze test and on expression of mRNA of proinflammatory cytokines and BDNF in different brain structures. Relevance of this study is related to poor understanding of the effects of acute and chronic stress on manifestation of cognitive brain functions, as well as ambiguity of the literature data on the effects of both stresses on hypothalamic pituitary axis and expression of the proinflammatory cytokine genes. In the experiments with rats, acute lipopolysaccharide (LPS)-induced stress improved learning in the Morris water maze. For the period of learning, the rats under acute stress swam on average less distance to reach a hidden platform, spent less time in the peripheral zone of the pool (thigmotaxis), and had low speed compared to the control animals and to the group of rats under chronic LPS-induced stress. In the test without a platform in the pool there were no significant differences between the groups on the time spent in the platform quadrant and distance swum. Acute stress caused substantial increase of the TNF-α and IL-1β mRNA concentrations in the hippocampus and amygdala, but not in the frontal lobe in comparison with the control animals. Although chronic stress increased the levels of the TNF-α and IL-1β mRNA in the amygdala and hippocampus compared to the control groups, significance between the groups was only marginal and BDNF concentration did not differ from the control animals in any of the brain structures mentioned. Expression of the IL-6 mRNA only marginally increased in the amygdala of the animals under the acute LPS-induced stress and marginally decreased in the animals under chronic LPS stress in the hippocampus relative to the control groups. In total, the most pronounced molecular-biochemical changes occurred in the amygdala and hippocampus, where increase of the expression of the TNF-α and IL-1β interleukins mRNAs were observed in the animals under acute and chronic LPS-induced stress and no changes in the BDNF mRNA concentration were observed in the frontal lobe.

P21 facilitates chronic lung inflammation via epithelial and endothelial cells.

Cellular senescence is a stable state of cell cycle arrest that regulates tissue integrity and protects the organism from tumorigenesis. However, the accumulation of senescent cells during aging contributes to age-related pathologies. One such pathology is chronic lung inflammation. p21 (CDKN1A) regulates cellular senescence via inhibition of cyclin-dependent kinases (CDKs). However, its role in chronic lung inflammation and functional impact on chronic lung disease, where senescent cells accumulate, is less understood. To elucidate the role of p21 in chronic lung inflammation, we subjected p21 knockout (p21-/-) mice to repetitive inhalations of lipopolysaccharide (LPS), an exposure that leads to chronic bronchitis and accumulation of senescent cells. p21 knockout led to a reduced presence of senescent cells, alleviated the pathological manifestations of chronic lung inflammation, and improved the fitness of the mice. The expression profiling of the lung cells revealed that resident epithelial and endothelial cells, but not immune cells, play a significant role in mediating the p21-dependent inflammatory response following chronic LPS exposure. Our results implicate p21 as a critical regulator of chronic bronchitis and a driver of chronic airway inflammation and lung destruction.

Disturbances of phosphatidylcholines metabolism in major depressive disorder.

Major depressive disorder (MDD) is a common neuropsychiatry disorder with high prevalence and recurrence rate, but the misdiagnosis rate is inevitable due to the shortage of objective laboratory-based diagnostic criteria. This study is focused on the disturbance of lipid metabolism, providing potential biomarkers for diagnosing. Lipid metabolism-related molecules in plasma of 42 drug-naïve MDD patients and 49 healthy people were measured by liquid chromatography-mass spectrometry. Further to evaluate the diagnostic values of changed metabolites, these molecules were evaluated by the receiver operating characteristic curve. Based on the significant role of phosphatidylcholine (PC) disturbance in depression, oxidization of PCs, oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC), IL-8 and caspase-3 in hippocampus, and serum of chronic lipopolysaccharide (cLPS) depression mice were detected by ELISA. Compared with healthy control, MDD patients expressed higher 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (16:0-16:0 PC, DPPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0-20:4 PC, PAPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-18:0 PC), glycocholic acid, taurocholic acid, glycoursodeoxycholic acid, and chenodeoxycholic acid glycine conjugate, and lower 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC 20:0). The 16:0-20:4 PC showed the great diagnostic value for MDD with an area under the curve (AUC) of 0.9519, and combination of 16:0 PC, 16:0-18:0 PC, and 16:0-20:4 PC exhibited the highest diagnostic value with AUC of 0.9602. OxPAPC was certified increase in hippocampus and serum of cLPS depression mice, which further supported PCs disorder participated in depression. This research offers 16:0-20:4 PC as the latent diagnostic indicator for MDD and hints the important role of PCs in depression.

Microbiota dysbiosis caused by dietetic patterns as a promoter of Alzheimer's disease through metabolic syndrome mechanisms.

Microbiota dysbiosis and metabolic syndrome, consequences of a non-adequate diet, generate a feedback pathogenic state implicated in Alzheimer's disease development. The lower production of short chain fatty acids (SCFAs) under dysbiosis status leads to lipid homeostasis deregulation and decreases Angptl4 release and AMPK activation in the adipose tissue, promoting higher lipid storage (adipocyte hypertrophy) and cholesterol levels. Also, low SCFA generation reduces GPR41 and GPR43 receptor activation at the adipose tissue (increasing leptin release and leptin receptor resistance) and intestinal levels, reducing the release of GLP-1 and YPP. Therefore, lower satiety sensation and energy expenditure occur, promoting a weight gaining environment mediated by higher food intake and lipid storage, developing dyslipemia. In this context, higher glucose levels, together with higher free fatty acids in the bloodstream, promote glycolipotoxicity, provoking a reduction in insulin released, insulin receptor resistance, advanced glycation products (AGEs) and type 2 diabetes. Intestinal dysbiosis and low SCFAs reduce bacterial biodiversity, increasing lipopolysaccharide (LPS)-producing bacteria and intestinal barrier permeability. Higher amounts of LPS pass to the bloodstream (endotoxemia), causing a low-grade chronic inflammatory state characterized by higher levels of leptin, IL-1β, IL-6 and TNF-α, together with a reduced release of adiponectin and IL-10. At the brain and neuronal levels, the generated insulin resistance, low-grade chronic inflammation, leptin resistance, AGE production and LPS increase directly impact the secretase enzymes and tau hyperphosphorylation, creating an enabling environment for β-amyloid senile plaque and tau tangled formations and, as a consequence, Alzheimer's initiation, development and maintenance.

Increased Expression and Activity of Brain Cortical cPLA2 Due to Chronic Lipopolysaccharide Administration in Mouse Model of Familial Alzheimer's Disease.

Cytosolic phospholipase A2 (cPLA2) is an enzyme regulating membrane phospholipid homeostasis and the release of arachidonic acid utilized in inflammatory responses. It represents an attractive target for the treatment of Alzheimer's disease (AD). Previously, we showed that lipopolysaccharide (LPS)-induced systemic inflammation caused abnormal lipid metabolism in the brain of a transgenic AD mouse model (APdE9), which might be associated with potential changes in cPLA2 activity. Here, we investigated changes in cPLA2 expression and activity, as well as the molecular mechanisms underlying these alterations due to chronic LPS administration in the cerebral cortex of female APdE9 mice as compared to saline- and LPS-treated female wild-type mice and saline-treated APdE9 mice. The study revealed the significant effects of genotype LPS treatment on cortical cPLA2 protein expression and activity in APdE9 mice. LPS treatment resulted in nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) activation in the cortex of APdE9 mice. The gene expressions of inflammation markers Il1b and Tnfa were significantly elevated in the cortex of both APdE9 groups compared to the wild-type groups. The study provides evidence of the elevated expression and activity of cPLA2 in the brain cortex of APdE9 mice after chronic LPS treatment, which could be associated with NFkB activation.

Chronic lipopolysaccharide impairs motivation when delivered to the ventricles, but not when delivered peripherally in male rats

Increased neuroinflammation relative to controls is observed in major depression. Moreover, depressive disorders are significantly elevated in conditions which increase neuroinflammation (e.g., brain injury, Parkinson's disease, Alzheimer's disease). To better understand the relationship between neuroinflammation and depression, additional research is needed. The current set of studies made use of the progressive ratio (PR) task in male rats, a stable measure of motivation which can be evaluated daily and thus is ideally suited for examining a potential role for chronic neuroinflammation in depressive-like behavior. Lipopolysaccharide (LPS) was used to induce an inflammatory response. Experiment 1 confirmed prior acute LPS administration experiments for sensitivity of the PR task, with a large effect at 2 mg/kg, a partial effect at 1 mg/kg, and no effect at 0.5 mg/kg. Experiment 2 evaluated a dose-response of continuous s.c. LPS infusion but found no significant elevation in brain cytokines after 14 days at any doses of 0.1, 0.5, 1, or 2 mg/kg/week. Experiment 3 assessed motivation during continuous s.c. infusion of a large 5 mg/kg/week LPS dose and found no significant impairments in motivation, but transient decreases in rates of lever pressing (i.e., only motoric deficits). Experiment 4 measured motivation during continuous ICV infusion of 10.5 μg/kg/week LPS and found significantly decreased motivation without changes to rates of lever pressing (i.e., only motivational deficits). Together these results suggest that the PR task is efficient for evaluating models of chronic inflammation, and that the adaptive response to chronic LPS exposure, even when delivered centrally, may necessitate alternative strategies for generating long-term neuroinflammation.

Upregulation of Circ_0035266 Contributes to the Malignant Progression of Inflammation-Associated Malignant Transformed Cells Induced by Tobacco-Specific Carcinogen NNK.

Cigarette smoking-induced chronic inflammation has been considered a vital driver of lung tumorigenesis. The compounds 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen, and lipopolysaccharide (LPS), an inflammatory inducer, are important components of tobacco smoke which have been implicated in inflammation-driven carcinogenesis. However, the biological effects and underlying mechanisms of LPS-mediated inflammation on NNK-induced tumorigenesis are still unclear. In this study, BEAS-2B human bronchial epithelial cells were exposed to NNK, LPS or both, for short- or long-term periods. We found that acute LPS exposure promoted the secretion of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-6 in NNK-treated BEAS-2B cells. In addition, chronic LPS exposure facilitated the NNK-induced malignant transformation process by promoting cell proliferation, cell cycle alteration, migration, and clonal formation. Previously, we determined that circular RNA circ_0035266 enhanced cellular inflammation in response to NNK + LPS by sponging miR-181d-5p and regulating expression of its downstream target DEAD-Box Helicase 3 X-Linked (DDX3X). Here, we found that knockdown of circ_0035266 or DDX3X led to a remarkable inhibition of the proliferation, cell cycle progression, and migration of NNK + LPS-transformed BEAS-2B cells, whereas overexpression of these genes produced the opposite effects, indicating the oncogenic roles of circ_0035266 and DDX3X in the malignant progression of chronic inflammation-driven malignant transformed cells. Moreover, the regulatory relationships among circ_0035266, miR-181d-5p, and DDX3X were further confirmed using a group of lung cancer tissues. Conclusively, our findings provide novel insights into our understanding of inflammation-driven tumorigenesis using a cellular malignant transformation model, and indicate a novel tumor-promoting role for circ_0035266 in chemical carcinogenesis.