Administration Of Zymosan Research Articles

Myeloperoxidase-deficient mice exposed to Zymosan exhibit severe neutrophilia and anemia with enhanced granulopoiesis and reduced erythropoiesis, accompanied by pulmonary inflammation

We previously reported that myeloperoxidase-deficient (MPO-/-) mice develop more severe neutrophil-rich lung inflammation than wild-type mice following intranasal Zymosan administration. Interestingly, we found that these mutant mice with severe lung inflammation also displayed pronounced neutrophilia and anemia, characterized by increased granulopoiesis and decreased erythropoiesis in the bone marrow, compared to wild-type mice. This condition was associated with higher concentrations of granulocyte-colony stimulating factor (G-CSF) in both the lungs and serum, a factor known to enhance granulopoiesis. Neutrophils accumulating in the lungs of MPO-/- mice produced greater amounts of G-CSF than those in wild-type mice, indicating that they are a significant source of G-CSF. In vitro experiments using signal transduction inhibitors and Western blot analysis revealed that MPO-/- neutrophils express higher levels of G-CSF mRNA in response to Zymosan, attributed to the upregulation of the IκB kinase/nuclear factor (NF)-κB pathway and the extracellular-signal-regulated kinase/NF-κB pathway. These findings highlight MPO as a critical regulator of granulopoiesis and erythropoiesis in inflamed tissues.

Characterization of two tripartite motif-containing genes from Asian Seabass Lates calcarifer and their expression in response to virus infection and microbial molecular motifs.

We identified two tripartite motif (TRIM) genes, LcTRIM21 and LcTRIM39, from the Asian Seabass Lates calcarifer, and examined their responses to experimental betanodavirus infection and stimulation with microbial pathogen-associated molecular patterns. Genes encoding LcTRIM21 and LcTRIM39 were identified, cloned, and sequenced from the Asian Seabass. We analyzed the sequence using a variety of bioinformatics tools to determine protein structure, localization, and establish a phylogenetic tree. By using quantitative real-time PCR, we analyzed expression profiles of the LcTRIM21 and LcTRIM39 genes in response to betanodavirus challenge as well as molecular pathogen-associated molecular patterns like poly(I:C) and Zymosan A. The tissue distribution pattern of these genes was also examined in healthy animals. Asian Seabass homologues of the TRIM gene, LcTRIM21 and LcTRIM39, were cloned, both encoding proteins with 547 amino acids. LcTRIM21 is predicted to have an isoelectric point of 6.32 and a molecular mass of 62.11 kilodaltons, while LcTRIM39 has an isoelectric point of 5.57 and a molecular mass of 62.11 kilodaltons. LcTRIM21 and LcTRIM39 homologues were predicted to be localized in cytoplasm by in silico protein localization. Structurally, both proteins contain an N-terminal really interesting new gene (RING) zinc-finger domain, B-box domain, coiled-coil domain and C-terminal PRY/SPRY domain. Most tissues and organs examined showed constitutive expression of LcTRIM21 and LcTRIM39. Upon poly(I:C) challenge or red-spotted grouper nervous necrosis virus infection, LcTRIM21 and LcTRIM39 mRNA expression was significantly upregulated, suggesting that they may play a critical antiviral role against fish viruses. LcTRIM21 and LcTRIM39 expression were also upregulated by administration of the glucan Zymosan A. The TRIM-containing gene is an E3 ubiquitin ligase that exhibits antiviral activity by targeting viral proteins via proteasome-mediated ubiquitination. TRIM proteins can be explored for the discovery of antivirals and strategies to combat diseases like viral nervous necrosis, that threaten seabass aquaculture.

Complement terminal pathway inhibition reduces peritoneal injuries in a rat peritonitis model.

Peritonitis and the resulting peritoneal injuries are common problems that prevent long-term peritoneal dialysis (PD) therapy in patients with end-stage kidney diseases. Previously, we have analyzed the relationship between the complement system and progression of peritoneal injuries associated with PD, particularly focusing on the early activation pathways and effects of the anaphylatoxins. We here utilized a novel mAb 2H2 that blocks assembly of the membrane attack complex (MAC) to investigate roles of the complement terminal pathway in PD-associated peritoneal injury. We intraperitoneally injected mAb 2H2 anti-C5b-7 (2.5 or 5 mg/rat) once or twice over the five-day course of the experiment to investigate the effects of inhibiting formation of MAC in a fungal rat peritonitis model caused by repeated intraperitoneal administration of zymosan after methylglyoxal pretreatment (Zy/MGO model). Rats were sacrificed on day 5 and macroscopic changes in both parietal and visceral peritoneum evaluated. Peritoneal thickness, the abundance of fibrinogen and complement C3 and MAC deposition in tissue and accumulation of inflammatory cells were pathologically assessed. The results showed that mAb 2H2, but not isotype control mAb, reduced peritoneal thickness and accumulation of inflammatory cells in a dose and frequency-dependent manner in the Zy/MGO model. These effects were accompanied by decreased C3, MAC, and fibrinogen deposition in peritoneum. In conclusion, in the rat Zy/MGO model, complement terminal pathway activation and MAC formation substantially contributed to development of peritoneal injuries, suggesting that MAC-targeted therapies might be effective in preventing development of peritoneal injuries in humans.

Zymosan-Induced Murine Peritonitis Is Associated with an Increased Sphingolipid Synthesis without Changing the Long to Very Long Chain Ceramide Ratio.

Sphingolipids are key molecules in inflammation and defense against pathogens. Their role in dectin-1/TLR2-mediated responses is, however, poorly understood. This study investigated the sphingolipidome in the peritoneal fluid, peritoneal cells, plasma, and spleens of mice after intraperitoneal injection of 0.1 mg zymosan/mouse or PBS as a control. Samples were collected at 2, 4, 8, and 16 h post-injection, using a total of 36 mice. Flow cytometry analysis of peritoneal cells and measurement of IL-6, IL-1β, and TNF-α levels in the peritoneal lavages confirmed zymosan-induced peritonitis. The concentrations of sphingoid bases, dihydroceramides, ceramides, dihydrosphingomyelins, sphingomyelins, monohexosylceramides, and lactosylceramides were increased after zymosan administration, and the effects varied with the time and the matrix measured. The greatest changes occurred in peritoneal cells, followed by peritoneal fluid, at 8 h and 4 h post-injection, respectively. Analysis of the sphingolipidome suggests that zymosan increased the de novo synthesis of sphingolipids without change in the C14-C18:C20-C26 ceramide ratio. At 16 h post-injection, glycosylceramides remained higher in treated than in control mice. A minor effect of zymosan was observed in plasma, whereas sphinganine, dihydrosphingomyelins, and monohexosylceramides were significantly increased in the spleen 16 h post-injection. The consequences of the observed changes in the sphingolipidome remain to be established.

Zymosan Particle-Induced Hemodynamic, Cytokine and Blood Cell Changes in Pigs: An Innate Immune Stimulation Model with Relevance to Cytokine Storm Syndrome and Severe COVID-19.

Hemodynamic disturbance, a rise in neutrophil-to-lymphocyte ratio (NLR) and release of inflammatory cytokines into blood, is a bad prognostic indicator in severe COVID-19 and other diseases involving cytokine storm syndrome (CSS). The purpose of this study was to explore if zymosan, a known stimulator of the innate immune system, could reproduce these changes in pigs. Pigs were instrumented for hemodynamic analysis and, after i.v. administration of zymosan, serial blood samples were taken to measure blood cell changes, cytokine gene transcription in PBMC and blood levels of inflammatory cytokines, using qPCR and ELISA. Zymosan bolus (0.1 mg/kg) elicited transient hemodynamic disturbance within minutes without detectable cytokine or blood cell changes. In contrast, infusion of 1 mg/kg zymosan triggered maximal pulmonary hypertension with tachycardia, lasting for 30 min. This was followed by a transient granulopenia and then, up to 6 h, major granulocytosis, resulting in a 3-4-fold increase in NLR. These changes were paralleled by massive transcription and/or rise in IL-6, TNF-alpha, CCL-2, CXCL-10, and IL-1RA in blood. There was significant correlation between lymphopenia and IL-6 gene expression. We conclude that the presented model may enable mechanistic studies on late-stage COVID-19 and CSS, as well as streamlined drug testing against these conditions.

The Impact of Avpr1a Expression and Host-Microbiome Interactions on Visceral Hypersensitivity

Visceral hypersensitivity (VH) is commonly cited as a major driver of chronic abdominal pain in "functional" gastrointestinal disorders (e.g., irritable bowel syndrome) where persistent and/or recurrent abdominal pain is the primary unifying symptom regardless of any alterations in bowel habits. The complexity of VH is in part influenced by genetic factors and individual differences in gut microbiome composition, yet specific mechanisms that generate VH remain incompletely understood. Correspondingly, current treatments to primarily focus on symptom management rather than targeting physiological mechanisms responsible for generating VH. We have begun to examine the role of genetic susceptibility and microbiome response dynamics in VH development using a preclinical model of intracolonic zymosan (ZYM) administration in which there are strain differences to VH susceptibility. Preliminary data reveals differential susceptibility between ZYM-induced VH in two closely related C57BL/6 sub strains, one from Taconic Biosciences (C57BL/6NTac) and the other from Jackson Laboratory (C57BL/6J). We have identified a VH candidate gene that encodes the arginine-vasopressin receptor 1A (AVPR1A) protein. We have further observed dynamic strain differences in the location and composition of the gut microbiome in response to ZYM corresponding to VH susceptibility. Ongoing studies are focused on teasing apart the potential bidirectional relationship(s) between genetic susceptibility and host-microbiome interactions in the etiology of VH. Identifying underlying mechanisms that drive VH would provide novel targets for pharmacological intervention and decrease reliance on opioids, which are prescribed at a significantly higher rate to patients who report abdominal pain with no accompanying structural disease. Grant support from R21 NS104789/NS/NINDS (KMB), R03 NS096454/NS/NINDS (KMB), Rita Allen Foundation Award in Pain (KMB), P20GM103418 (EEY and KMB), and a K-INBRE recruitment startup package.

Anti-PCSK9 monoclonal antibody attenuates high-fat diet and zymosan-induced vascular inflammation in C57BL/6 mice by modulating TLR2/NF-ƙB signaling pathway.

Objective(s):Excess intake of a high-fatty diet (HFD) together with zymosan administration mediates vasculitis response which leads to impaired serum lipid levels and causes arterial stiffness. In the development of new cholesterol-lowering medications, PCSK9 inhibitor (proprotein convertase subtilisin/kexin type 9) is an emerging therapeutic. The goal of the present study was to see whether anti-PCSK9 mAb1 might prevent vasculitis in C57BL/6 mice by blocking TLR2/NF-B activation in HFD and Zymosan-induced vasculitis. Materials and Methods:Protein-protein molecular docking was performed to validate the binding affinity of anti-PCSK9 mAb1 against TLR2. Under the experimental study, mice were randomly allocated to the following groups: Group I: standard mice diet (30 days) + Zymosan vehicle (sterile PBS solution of 5mg/ml on 8th day); Group II: HFD (30 days) + Zymosan ( single IP dose 80 mg/kg on day 8th); Group III: HFD+Zymosan + anti-PCSK9 mAb1 (6 mg/kg, s.c. on 10th and 20th days); Group IV: HFD+Zymosan+anti-PCSK9 mAb1 (10 mg/kg, s.c. on 10th and 20th days).Results:In comparison with the low dose of anti-PCSK9 mAb1 (6 mg/kg), the high dose of anti-PCSK9 mAb1 (10 mg/kg) together with HFD and Zymosan inhibited vasculitis more effectively by decreasing aortic TLR2 and NF-B levels, reducing serum TNF- and IL-6, and up-regulating liver LDLR levels, which down-regulated serum LDL-C and improved serum lipids levels. Histopathological studies showed that anti-PCSK9 mAb1 treatment reduced plaque accumulation in the aorta of mice.Conclusion:These findings indicate that anti-PCSK9 mAb1 has therapeutic potential in reducing HFD and Zymosan-induced vascular inflammation.

Calceolarioside A, a Phenylpropanoid Glycoside from Calceolaria spp., Displays Antinociceptive and Anti-Inflammatory Properties.

Phenylpropanoid glycosides are a class of natural substances of plant origin with interesting biological activities and pharmacological properties. This study reports the antinociceptive and anti-inflammatory effects of calceolarioside A, a phenylpropanoid glycoside previously isolated from various Calceolaria species. In models of acute nociception induced by thermal stimuli, such as the hot plate and tail flick test, calceolarioside administered at doses of 1, 5, and 10 μg in the left cerebral ventricles did not modify the behavioral response of mice. In an inflammatory based persistent pain model as the formalin test, calceolarioside A at the high dose tested (100 μg/paw) reduced the licking activity induced by formalin by 35% in the first phase and by 75% in the second phase of the test. In carrageenan-induced thermal hyperalgesia, calceolarioside A (50 and 100 μg/paw) was able to significantly reverse thermal hyperalgesia induced by carrageenan. The anti-inflammatory activity of calceolarioside A was then assessed using the zymosan-induced paw edema model. Calceolarioside A (50 and 100 μg/paw) induced a significant reduction in the edema from 1 to 4 h after zymosan administration. Measuring IL-6, TNFα, and IL-1β pro-inflammatory cytokines released from LPS-stimulated THP-1 cells, calceolarioside A in a concentration-dependent manner reduced the release of these cytokines from THP-1 cells. Taken together, our results highlight, for the first time, the potential and selective anti-inflammatory properties of this natural-derived compound, prompting its rationale use for further investigations.

Neonatal Bladder Inflammation Results in Adult Female Mouse Phenotype With Increased Frequency and Nociceptive Responses to Bladder Filling.

Bladder pain and hypersensitivity to bladder filling are clinically common, but animal models examining syndromes with these features are limited. A rat model of bladder hypersensitivity produced by neonatal bladder inflammation (NBI) has been reported to have many of the clinical features of bladder pain syndromes. The present study sought to determine whether similar hypersensitivity might be induced by NBI in mice. Female C57BL6/J mice had NBI induced on postnatal days P12-14 by the intravesical administration of zymosan. As adults (12–14 weeks of age), the mice were examined for hypersensitivity of their bladders as: spontaneous voiding and evoked cystometrograms at baseline, and visceromotor responses (VMRs) to urinary bladder distension (UBD) following a secondary insult (either repeated bladder inflammation or acute stress induced by footshock). Mice that experienced NBI demonstrated hypersensitivity, when compared with control mice, manifested as increased spontaneous voiding, increased frequency of evoked voids during intravesical saline infusion, and increased vigor of VMRs to UBD following either acute bladder inflammation or acute stress. This recapitulates the hallmark features of clinical painful bladder disorders and suggest utility of this murine model for the study of these disorders while allowing methodological expansion into well-established genetic and immunological models.

Oral administration of eucalyptol reduces cell migration and pain-like behavior in zymosan-induced arthritis mice

Rheumatoid arthritis (RA) is an inflammatory disease that utilizes nonbiologic and biologic drugs for appropriate disease management. However, high cost, adverse effects, reduced effectiveness, and risk of infection have stimulated the search for safer and more efficacious therapeutic strategies. In the present study, we aimed to evaluate the anti-inflammatory and analgesic properties of eucalyptol in an experimental model of arthritis. Mice were administered zymosan or saline intra-articularly. One hour before the zymosan administration, the mice were treated with oral eucalyptol (200-400 mg/kg) and vehicle. Cell influx, neutrophils, lymphocytes, and monocytes were measured in joint exudates. Joint pain was assessed using paw-pressure tests. Orally administered eucalyptol (200 and 400 mg/kg) significantly reduced cell influx, as well as neutrophils, lymphocytes, and monocytes, when compared with the control. Eucalyptol at a dose of 400 mg/kg significantly reversed joint pain and demonstrated analgesic activity (60%); however, 200 mg/kg failed to alter joint pain. These results indicate that oral eucalyptol promotes anti-inflammatory and analgesic activity in mice subjected to zymosan-induced arthritis.

Mechanisms of action of radon therapy on cytokine levels in normal mice and rheumatoid arthritis mouse model.

The typical indication of radon therapy is rheumatoid arthritis. Although there are several reports that radon therapy has regulation effects on Th17 cells, there has been no study reporting that radon inhalation affects the immune balance among Th1, Th2, and Th17. The purpose of this study is to examine the cytokine changes after radon inhalation. BALB/c mice inhaled radon at 2,000 Bq/m3 for 2 or 4 weeks. SKG/Jcl mice inhaled radon at 2,000 Bq/m3 for 4 weeks after zymosan administration. The results showed that radon inhalation for 4 weeks activated the immune response of Th1, Th2, and Th17. Moreover, the balance among them was not lost by radon inhalation. Radon inhalation for 4 weeks decreased superoxide dismutase activity and increased catalase activity in spleen. These findings suggest that an imbalance of oxidative stress may contribute to activate the immune response. Although zymosan administration activated Th17 immune response and decreased Th1 and Th2 immune response in SKG/Jcl mice, most cytokines related to Th1, Th2, and Th17 approached the normal level by radon inhalation. These findings suggested that radon inhalation has a different action between SKG/Jcl mice and normal BABL/c mice. This may indicate that radon inhalation has an immunomodulation function.

Detailed Characterization of Brain Dysfunction in a Long-Term Rodent Model of Critical Illness.

Critical illness encompasses a wide spectrum of life-threatening clinical conditions requiring intensive care. Our objective was to evaluate cognitive, inflammatory and cellular metabolism alterations in the central nervous system in an animal model of critical illness induced by zymosan. For this Wistar rats that were divided into Sham and zymosan. Zymozan was administered once intraperitoneally (30g/100g body weight) diluted in mineral oil. The animals were submitted to behavioral tests of octagonal maze, inhibitory avoidance and elevated plus maze. Brain structures (cortex, prefrontal and hippocampus) were removed at 24h, 4, 7 and 15days after zymosan administration for analysis of cytokine levels (TNF-α, IL-1b, IL-6 and IL-10), oxidative damage and oxygen consumption. Zymosan-treated animals presented mild cognitive impairment both in aversive (inhibitory avoidance) and non-aversive (octagonal maze) tasks by day 15. However, they did not show increase in anxiety (elevated-plus maze). The first neurochemical alteration found was an increase in brain pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) at day 4th in the hippocampus. In cortex, a late (7 and 15days) increase in TNF-α was also noted, while the anti-inflammatory cytokine IL-10 decrease from 4 to 15days. Oxygen consumption was decreased in the hippocampus and pre-frontal, but not cortex, only at 7days. Additionally, it was observed a late (15days) increase in oxidative damage parameters. This characterization of brain dysfunction in rodent model of critical illness reproduces some of the alterations reported in humans such neuropsychiatric disorders, especially depression, memory loss and cognitive changes and can add to the nowadays used models.

Repeated Stimulation of Toll-Like Receptor 2 and Dectin-1 Induces Chronic Pancreatitis in Mice Through the Participation of Acquired Immunity

Stimulation of Toll-like receptor 3 (TLR3) induces autoimmune-mediated pancreatitis in susceptible mice, whereas stimulation of TLR4 causes nonautoimmune-mediated pancreatitis. However, the effects of TLR2 stimulation on the pancreas are unknown. We investigated the role of TLR2 stimulation on pancreatic damage by repeatedly stimulating mice with TLR2 ligands. Wild-type (WT) and interleukin 10-deficient (IL-10-knockout (KO)) mice were administered zymosan and lipoteichoic acid (LTA) intraperitoneally at various doses twice weekly for 4weeks. Syngeneic T-cell-deficient mice, B-cell-deficient mice, recombination activating gene 2-deficient (RAG2-KO) mice and RAG2-KO mice that had been reconstituted with CD4+ or CD8+ T cells isolated from WT mice were treated with zymosan similarly. Mice were killed, the severity of pancreatitis was graded histologically, and serum cytokine levels were measured. Repeated administration of zymosan induced pancreatitis dose dependently in both WT and IL-10-KO mice. Administration of LTA induced pancreatitis only in IL-10-KO mice. Adoptive transfer of splenocytes obtained from IL-10-KO mice with pancreatitis did not cause pancreatitis in recipient RAG2-KO mice. Pancreatitis was scarcely observed in RAG2-KO mice and was attenuated in T-cell-deficient and B-cell-deficient mice compared with WT mice. A single administration of zymosan significantly increased the serum level of monocyte chemoattractant protein 1 (MCP-1) in WT mice. Repeated stimulation of TLR2 and dectin-1 induced nonautoimmune-mediated pancreatitis in mice. Participation of acquired immunity seems to play an important role in the pathogenesis of pancreatitis in association with the increase in serum MCP-1 level.

Role of TRPM8 in switching between fever and hypothermia in adult mice during endotoxin-induced inflammation

Transient receptor potential melastatin 8 (TRPM8) functions in the sensing of noxious and innocuous colds; however, its significance in pathogen-induced thermoregulation remains unclear. In the present study, we investigated the role of TRPM8 in the regulation of endotoxin-induced body temperature control. The peripheral administration of low-dose lipopolysaccharide (LPS) at 50 ​μg/kg generated fever in wild-type (WT) mice, whereas it caused hypothermia in TRPM8 knockout (KO) animals. LPS-induced sickness responses such as decrease in body weight, and food and water intake were not different between WT and TRPM8 KO mice. TRPM8 KO mice exhibited more severe hypothermia and lower locomotor activity following the peripheral administration of high-dose LPS at 5 ​mg/kg compared with WT ones. An intracerebroventricular (i.c.v.) injection of either LPS at 3.6 ​μg/kg or interleukin-1β at 400 ​ng/kg elicited hypothermia in TRPM8 KO mice, in contrast to fever in WT animals. The peripheral administration of zymosan at 3 ​mg/kg also induced hypothermia in contrast to fever in WT mice. An i.c.v. injection of prostaglandin E2 at 16 or 160 ​nmol/kg induced normal fever in both WT and TRPM8 KO mice. Infrared thermography showed significant decline of the interscapular skin temperature that estimates temperature of the brown adipose tissue, regardless of no alteration of its temperature in WT animals. Fos immunohistochemistry showed stronger Fos activation of hypothalamic thermoregulation-associated nuclei in TRPM8 KO mice compared with WT animals following the peripheral administration of low-dose LPS. Therefore, the present study indicates that TRPM8 is necessary for switching between fever and hypothermia during endotoxin-induced inflammation.

PSB 603 – a known selective adenosine A2B receptor antagonist – has anti-inflammatory activity in mice

A2B adenosine receptors are present in a wide spectrum of tissues, especially on cells of the immune system. Since these particular receptors have the lowest, of all adenosine receptor subtypes, affinity for adenosine they are believed to play a special role in immunological processes associated with elevated adenosine levels such as inflammation.The aim of this preliminary study was to determine the potential anti-inflammatory properties of compound PSB-603, a potent and selective adenosine A2B receptor antagonist, in two different experimental models of local and systemic inflammation.In a model of inflammation induced by local carrageenan administration paw edema was measured using a pletysmometer. Additionally, levels of C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and reactive oxygen species (ROS) were determined in the inflamed paw. Using the mouse model of peripheral inflammation induced by intraperitoneal (ip) administration of zymosan A, the influence of the A2B antagonist on the infiltration of neutrophils into the peritoneum and its effect on the plasma levels of CRP, TNF-α, and IL-6 were investigated.The results showed that PSB-603 administered at a dose of 5 mg/kg b.w. ip significantly reduced inflammation in both tested models. Particularly, it significantly decreased levels of the inflammatory cytokines IL-6, TNF-α and of ROS in the inflamed paw and reduced inflammation of the peritoneum by significantly decreasing the infiltration of leukocytes. Additionally, in the latter model, no statistically significant difference was observed in the CRP level between the control group without inflammation and the group which has been treated with the PSB-603 compound. Thus, the results may indicate the anti-inflammatory activity of adenosine A2B receptor antagonists in two different models of inflammation.

Depletion of microglia and macrophages with clodronate liposomes attenuates zymosan-induced Fos expression and hypothermia in the adult mouse

Toll-like receptor 2 (TLR2) recognizes a wide range of microbial molecules and plays critical roles in the initiation of innate immune responses. In the present study, we aimed to investigate whether the depletion of microglia and macrophages with clodronate liposomes (Clod-Lips) attenuates the activation of mouse brain circuits for TLR2-mediated inflammation and hypothermia. The peripheral administration of the TLR2 agonist zymosan induced nuclear factor-κB activation in microglia and macrophages and Fos expression in astrocytes/tanycytes and neurons in the circumventricular organs (CVOs). The depletion of microglia and macrophages with Clod-Lips markedly decreased zymosan-induced Fos expression in astrocytes/tanycytes and neurons in the CVOs. The treatment with Clod-Lips significantly attenuated zymosan-induced hypothermia. These results indicate that microglia and macrophages in the CVOs participate in the initiation and transmission of inflammatory responses after the peripheral administration of zymosan.

MP28-12 EFFECT OF A BIFUNCTIONAL MU/DELTA OPIOID RECEPTOR LIGAND ON MECHANOSENSITIVE PELVIC NERVE AFFERENT FIBERS INNERVATING THE URINARY BLADDER OF RATS

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I (MP28)1 Apr 2020MP28-12 EFFECT OF A BIFUNCTIONAL MU/DELTA OPIOID RECEPTOR LIGAND ON MECHANOSENSITIVE PELVIC NERVE AFFERENT FIBERS INNERVATING THE URINARY BLADDER OF RATS Maia Terashvili*, Pradeep Kannampalli, Bidyut K. Medda, Banani Banerjee, Christopher W. Cunningham, and Jyoti N. Sengupta Maia Terashvili*Maia Terashvili* More articles by this author , Pradeep KannampalliPradeep Kannampalli More articles by this author , Bidyut K. MeddaBidyut K. Medda More articles by this author , Banani BanerjeeBanani Banerjee More articles by this author , Christopher W. CunninghamChristopher W. Cunningham More articles by this author , and Jyoti N. SenguptaJyoti N. Sengupta More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000867.012AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Interstitial cystitis/painful bladder syndrome (IC/PBS) affects about 3-8 million women and 1-4 million men in the United States. There is an immediate need to develop medications that can effectively manage the severity of pain associated with IC/PBS. The conventional opioid therapy like morphine and its derivatives carry multiple adverse effects including tolerance, addiction, constipation and dependence. A bifunctional opioid ligand acts on two opioid receptors, but does not produce adverse CNS effects. This study investigates the analgesic effect of benzylideneoxymorphone (BOM), a bifunctional partial opioid ligand, which acts as partial agonist for mu-opioid receptors (MORs) and antagonist for delta-opioid receptors (DORs), in a rat model of cystitis-induced bladder pain. Due to unique pharmacological profile of BOM, we hypothesized that this compound would be effective in managing pain in IC/PBS patients without producing adverse CNS effects. METHODS: In our study, Inflammation was induced in female rats by intravesical administration of zymosan (5% in saline) for 3 days. On day 5, we investigated the inhibitory effect of different doses (1, 5 and 10mg/Kg, i.v.) of BOM on the responses of urinary bladder distension (UBD)-sensitive pelvic nerve afferent (PNA) fibers during bladder distension of non-inflamed naïve and cystitis rats. In addition, MORs expression in the lumbo-sacral (L6-S1) spinal cord was evaluated in Western Blot analysis experiments. RESULTS: The drug produced a dose-dependent inhibition of responses of UBD-sensitive PNA fibers in the lumbar 6 (L6) dorsal root from zymosan-induced cystitis rats. In contrast, in naïve non-inflamed rats BOM did not produce inhibition of responses of UBD-sensitive PNA afferents fibers. The Western Blot analysis of tissues from L6-S1 spinal cord exhibited marked upregulation of MORs protein expression of tissues from cystitis rats compared to that of non-inflamed naïve rats. CONCLUSIONS: These results indicate that BOM attenuates responses of UBD-sensitive PNA fibers of cystitis rats in contrast to non-inflamed rats, which could be due to altered expressions of MOR in the bladder innervating PNA afferent fibers. Therefore, BOM could be a leading compound as analgesic drug to manage pain associated with IC/PBS. In addition, the drug can be promising to manage chronic pelvic pain without adverse CNS effects. Source of Funding: NIH R01DK099201 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e424-e424 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Maia Terashvili* More articles by this author Pradeep Kannampalli More articles by this author Bidyut K. Medda More articles by this author Banani Banerjee More articles by this author Christopher W. Cunningham More articles by this author Jyoti N. Sengupta More articles by this author Expand All Advertisement PDF downloadLoading ...

Systemic microbial TLR2 agonists induce neurodegeneration in Alzheimer\u2019s disease mice

BackgroundAccumulating data suggest a central role for brain microglia in mediating cortical neuronal death in Alzheimer’s disease (AD), and for Toll-like receptor 2 (TLR2) in their toxic activation. Amyloid deposition in preclinical AD is associated with microglial activation but not directly with neurodegeneration. We examined in transgenic 5xFAD mice the hypothesis that systemic TLR2 agonists, derived from common infectious agents, may accelerate neurodegeneration in AD.MethodsMicrobial wall-derived TLR2 agonists zymosan and lipoteichoic acid were administered intraperitoneally or intracerebroventricularly to 7-month-old wild-type or 5xFAD mice. Immunofluorescent stainings were used to quantify cortical neurons and evaluate tissue reaction. Microglial activation was assessed using functional assays, RNA expression, and FACS analysis.ResultsRepeated low-dose systemic administration of zymosan or lipoteichoic acid killed cortical neurons in 5xFAD mice but not in wild-type mice. Direct CNS delivery of a selective TLR2 antagonist blocked the neurotoxicity of systemically administered zymosan, indicating that CNS TLR2 mediates this effect. Systemically administered zymosan crossed the disrupted blood-brain barrier in 5xFAD mice and entered brain parenchyma. By intracerebroventricular delivery, we found a dose- and exposure time-dependent acute neurotoxic effect of the microbial TLR2 agonist, killing cortical neurons. 5xFAD mice exhibited significantly increased vulnerability to TLR2 agonist-induced neuronal loss as compared to wild-type mice. Microbial TLR2-induced neurodegeneration was abolished by inhibiting microglia. The vulnerability of 5xFAD mice brains was mediated by an increase in number and neurotoxic phenotype of TLR2-expressing microglia.ConclusionsWe suggest that repeated exposure to microbial TLR2 agonists may facilitate neurodegeneration in AD by their microglial-mediated toxicity to the hyper-vulnerable environment of the AD brain.

NADPH Oxidase Controls Neutrophil Infiltration in the Lung in Early Response to Fungal Cell Walls Via LTB4

The leukocyte NADPH oxidase generates superoxide, the precursor to reactive oxygen species important for host defense and immunoregulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by serious bacterial and fungal infections and aberrant inflammation. Aspergillus pneumonia is frequent, and associated with pyogranulomatous inflammation. Of note, even sterile fungal cell walls elicit increased neutrophilic inflammation in CGD mice, demonstrating that that NADPH oxidase can limit inflammation independent of its antimicrobial effects. Leukotriene B4 (LTB4) is a potent inflammatory mediator, acting as a chemoattractant and activating other polymorphonuclear leukocyte (PMN) functions. PMN themselves are prominent source of LTB4, and sense LTB4 through its receptor BLT1, which stimulates additional LTB4 synthesis by autocrine and paracrine routes. This leads to feed forward amplification of LTB4 production locally and formation of PMN clusters. Ca2+ acts as an important second messenger in PMN, including regulation of LTB4 synthesis. Activated human CGD PMN have increased intracellular Ca2+ levels compared to PMN from healthy donors due to electrogenic effects of the NADPH oxidase (Geiszt M. et al JBC 1997). Intracellular Ca2+ overload in PMN is speculated to contribute to increased PMN pro-inflammatory activity, but specific pathways and in vivo relevance are not well defined. Therefore, we therefore investigated the production of LTB4 by murine CGD PMN and the role of LTB4 in PMN recruitment and lung hyperinflammation following pulmonary challenge with zymosan, a sterile fungal cell wall preparation. Zymosan-stimulated PMN from Cybb-null mice, a model for X-linked CGD, produced higher LTB4 in vitro compared to wild type (WT), even in the presence of SOD and catalase to counteract oxidative degradation. Higher LTB4 production correlated with higher levels of intracellular Ca2+ in stimulated X-CGD PMN. The increased LTB4 produced by X-CGD PMN was dependent on cell density and the LTB4 receptor BLT1, consistent with a feed-forward loop that amplified LTB4 production. Zymosan-stimulated X-CGD PMN also formed larger and more numerous clusters in vitro compared to WT PMN. Cluster formation was abrogated by inhibiting LTB4 synthesis with the 5-lipoxygenase (5-LO) inhibitor zileuton or by the BLT1 receptor antagonist U75302, demonstrating that cluster formation was LTB4 and BLT1 dependent. We next examined whether LTB4 regulated the response to zymosan in the lung. Following intranasal administration of zymosan, X-CGD mice had higher PMN numbers in bronchoalveolar lavage (BAL), larger PMN foci by lung histology, and increased lung LTB4 as compared to WT mice. These differences were evident by 8 hr and progressed over the first 24 hr post-challenge, in contrast to WT mice where inflammation plateaued at 8 hr. Treatment with zileuton or U75302 just prior to zymosan significantly reduced lung PMN numbers for both WT and X-CGD mice at 8 hr post challenge, and lung PMN remained substantially reduced in X-CGD mice at 24 hrs. Moreover, delaying administration of zileuton to 8 hr after zymosan challenge also significantly decreased PMN inflammation in X-CGD but not WT mice at 24 hrs, indicating that ongoing synthesis of LTB4 otherwise continued to promote PMN recruitment in CGD mice. These results demonstrate that PMN production of LTB4 in response to fungal cell walls is limited by NADPH oxidase via its effects on intracellular Ca2+. In oxidase-deficient CGD mice, LTB4 plays a major role in driving excessive PMN recruitment to the lung in the early response to zymosan. This study is the first to implicate LTB4 in promoting neutrophilic lung inflammation in response to fungal cell walls in CGD, likely by an amplified feed-forward loop involving increased production of LTB4 by CGD PMN. LTB4 could be a potential therapeutic target to ameliorate CGD hyperinflammation. Disclosures No relevant conflicts of interest to declare.

Pyrogenic and neuroinflammatory properties of zymosan and its potential as an alternative to live yeast in antipyretic drug testing

Zymosan, an immunogenic cell wall extract of Saccharomyces cerevisiae has potential for use as an experimental pyrogen. However, the short-lived sickness responses noted with intraperitoneal and intra-articular administration of zymosan limits investigations on the long-term effectiveness of antipyretic drugs. Thus, there remains a need to establish an alternative route of zymosan administration that could induce long-lived fevers and inflammation. We injected male Sprague Dawley rats (250–300 g) subcutaneously with zymosan (30 or 300 mg/kg) or saline; n = 7–8. We measured core body temperature, cage activity, food intake and body mass for 24 h after injection. Blood and brain samples were collected at 2, 8, and 18 h after injection. Zymosan (300 mg/kg) induced fever, lethargy, and anorexia, which lasted for 24 h. Zymosan-induced sickness responses were accompanied by increased blood plasma levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α; activation of inflammatory transcription factors (nuclear factor (NF) for IL-6, signal transducer and activator of transcription (STAT)-3, and NF-κB) in the hypothalamus and circumventricular organs; and increased hypothalamic mRNA expression of TNF-α, IL-1β, and IL-6 and rate-limiting enzymes for prostaglandin synthesis. Our results confirm the suitability of subcutaneous administration of zymosan for screening antipyretic and anti-inflammatory drugs in rats.