IL-10 Knockout Mice Research Articles

Interleukin-6 trans-signaling contributes to chronic hypoxia-induced pulmonary hypertension.

Interleukin-6 (IL-6) is a pleotropic cytokine that signals through the membrane-bound IL-6 receptor (mIL-6R) to induce anti-inflammatory (“classic-signaling”) responses. This cytokine also binds to the soluble IL-6R (sIL-6R) to promote inflammation (“trans-signaling”). mIL-6R expression is restricted to hepatocytes and immune cells. Activated T cells release sIL-6R into adjacent tissues to induce trans-signaling. These cellular actions require the ubiquitously expressed membrane receptor gp130. Reports show that IL-6 is produced by pulmonary arterial smooth muscle cells (PASMCs) exposed to hypoxia in culture as well as the medial layer of the pulmonary arteries in mice exposed to chronic hypoxia (CH), and IL-6 knockout mice are protected from CH-induced pulmonary hypertension (PH). IL-6 has the potential to contribute to a broad array of downstream effects, such as cell growth and migration. CH-induced PH is associated with increased proliferation and migration of PASMCs to previously non-muscularized vessels of the lung. We tested the hypothesis that IL-6 trans-signaling contributes to CH-induced PH and arterial remodeling. Plasma levels of sgp130 were significantly decreased in mice exposed to CH (380 mmHg) for five days compared to normoxic control mice (630 mmHg), while sIL-6R levels were unchanged. Consistent with our hypothesis, mice that received the IL-6 trans-signaling-specific inhibitor sgp130Fc, a fusion protein of the soluble extracellular portion of gp130 with the constant portion of the mouse IgG1 antibody, showed attenuation of CH-induced increases in right ventricular systolic pressure, right ventricular and pulmonary arterial remodeling as compared to vehicle (saline)-treated control mice. In addition, PASMCs cultured in the presence of IL-6 and sIL-6R showed enhanced migration but not proliferation compared to those treated with IL-6 or sIL-6R alone or in the presence of sgp130Fc. These results indicate that IL-6 trans-signaling contributes to pulmonary arterial cell migration and CH-induced PH.

Abstract 4738: IL-17 induces myeloid-related stimulating factors by stromal cells

Abstract Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive cancer with a 5-year survival rate of ∼ 8%. An hallmark of PDA is the massive desmoplasia and the complex stromal-tumor interaction, regulated by a pletory of secreted factors. The role of IL-17 and T-helper 17 in cancer progression or anti-tumour immunity remain controversial. To assess the role of this molecule, we crossed genetically engineered mice (GEM) characterized by a spontaneous PDA development, with IL-17 knockout mice (GEM/IL-17 KO). The absence of IL-17 correlated with an increased pancreatic fibrosis, evaluated by second harmonic generation approach. The transcripts and secretome of Cancer Associated Fibroblasts (CAFs), obtained from tumors arose in both WT and IL-17 KO GEM, have been evaluated. More than 60 genes were differently modulated in the CAFs from GEM/IL-17 KO mice. Among them, GM-CSF, G-CSF, M-CSF, CXCL1 and 2 where strongly up-regulated in GEM/WT compared to GEM/IL-17 KO mice and significantly inhibited by the addition of the recombinant cytokine. The absence of those factors paralleled an increased Th1-skewed cytokine pattern released by CAF that affected T cell infiltration and response. Taken together, these results suggest that IL-17 does affect the tumor progression by promoting a CAFs-mediated release of cytokines involved in myeloid cells recruitment and suppression of an anti-tumoral immune response. This feature defines IL-17 as a potential target to shape the tumor microenvironment and favour immunotherapy success. Citation Format: Gianluca Mucciolo, Roberta Curto, Claudia Curcio, Cecilia Roux, Luca Vannucci, Francesco Novelli, Paola Cappello. IL-17 induces myeloid-related stimulating factors by stromal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4738.

Abnormal bone and cartilage metabolism could be antagonized by pulsed electromagnetic fields (PEMFS) and TNF-α and IL-6 gene knockouts in a similar mechanism

PEMFs, as a non-invasive method, could positively affect bone and cartilage metabolism. However, the bio-effect and underlying mechanisms remain poorly understood. The present study is designed to investigate the effect of PEMFs on osteoporotic bone and degenerative cartilage together with its potential molecular mechanisms in mice with different gene background. Twenty 12-week male and female wild-type (WT), TNFα knockout (TNFα −/− ) or IL6 knockout (IL6 −/− ) mice, respectively, were sham-operated (SHAM) or subjected to destabilization of the medial meniscus (DMM) and ovariectomy (OVX) surgeries. After surgeries, WT mice were equally assigned to the non-treatment and PEMFs groups. After surgeries, WT mice were equally assigned to the non-treatment and PEMFs groups. Mice in PEMFs group were subjected to daily 1-hour PEMFs exposure with 8 Hz, 3.8 mT (peak value). Then all mice were euthanized after 4 weeks. The surgical models of osteoporosis and osteoarthritis were proved successful evidenced by the analysis of micro-CT data and histological analysis. The bone loss and damaged cartilage were largely repaired by TNF-α and IL-6 gene knockout and partially inhibited by PEMFs exposure. Interestingly, no difference in Micro-CT data analysis was found between PEMFs group and gene knockouts, although a slight increase could be observed in TNFα −/− mice when compared to the PEMFs group. Negative effects on bone and cartilage were proved by testing key cytokines in anabolism and catabolism. PEMFs treatment and gene knockouts corrected the negative effects by targeting mediators in molecular pathways like Wnt and RANK in a similar way. PEMFs alleviated surgeries induced bone loss and cartilage degeneration by promoting anabolism and inhibiting catabolism possibly in a similar mechanism to TNF-α and IL-6 gene knockouts, which imply that TNF-α and IL-6 may become new potential targets for PEMFs in treating degenerative bone diseases.

Abstract 1102: IL-6/STAT3 activation in hepatocytes drives pro-metastatic niche formation in the liver

Abstract The liver is the most common site of metastasis in pancreatic ductal adenocarcinoma (PDAC). This metastatic tropism is dependent, at least in part, on the formation of a “pro-metastatic” niche that supports tumor cell seeding and colonization in the liver. However, mechanisms that direct the formation of this niche remain poorly understood. We show using the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) model of PDAC that pancreatic tumor development enhances the susceptibility of the liver to metastatic seeding by inducing recruitment of F4/80+ and Ly6G+ myeloid cells and fibrosis within the liver. 3' mRNA sequencing (QuantSeq) on RNA isolated from the liver of KPC mice versus control PC mice revealed that the liver produces a specific set of myeloid chemoattractants, particularly serum amyloid A1 and A2 (SAA1/2), early during PDAC development. In addition, gene set enrichment analysis (GSEA) on genes upregulated in the liver of KPC mice demonstrated a significant enrichment of the interleukin 6 (IL-6)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway. Consistent with this finding, phosphorylation of STAT3 was detected in 20-30% of F4/80+ myeloid cells and 80-90% of hepatocytes. A requirement for IL-6/STAT3 signaling in the formation of a pro-metastatic niche was determined by comparing the metastatic potential of wild type mice, Il-6 knockout (Il-6-/-) mice, and mice treated with anti-IL-6 receptor (IL-6R) antibody after orthotopic implantation of KPC-derived PDAC cells. Compared to wild type mice, the liver of Il-6-/- mice and mice treated with anti-IL-6R antibody was less susceptible to metastatic seeding and showed significantly less accumulation of myeloid cells, fibrosis, and production of SAA1/2 in the liver. We obtained similar results with mice that lack Stat3 specifically in hepatocytes (Stat3flox/flox Alb-Cre), demonstrating that IL-6/STAT3 signaling in hepatocytes is necessary for the formation of a pro-metastatic niche in the liver. Further, using Saa1/2 double knockout (Saa-/-) mice, we found that SAA1/2 production by hepatocytes was required for formation of the pro-metastatic niche in the liver and increased susceptibility to metastatic seeding. Patients with a history of liver metastasis also showed higher levels of SAA1/2 in the plasma compared to normal donors, and SAA overexpression was detected in hepatocytes in liver biopsy samples collected from PDAC patients. Collectively, our study reveals a novel role for hepatocytes in directing the formation of a pro-metastatic niche in the liver during PDAC development and identifies IL-6/STAT3/SAA1/2 signaling as a promising therapeutic target for prevention of metastasis in PDAC. Citation Format: Jae W. Lee, Stacy K. Thomas, Chad A. Komar, Whitney L. Gladney, Xia Hua, Dong Xin, Abraham Shaked, Mitesh J. Borad, Ramesh K. Ramanathan, Ailing Ji, Nancy R. Webb, Maria C. de Beer, Frederick C. de Beer, Paige M. Porrett, Gregory L. Beatty. IL-6/STAT3 activation in hepatocytes drives pro-metastatic niche formation in the liver [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1102.

In Vivo Validation Model of a Novel Anti-Inflammatory Scaffold in Interleukin-10 Knockout Mouse.

We fabricated anti-inflammatory scaffold using Mg(OH)2-incorporated polylactic acid-polyglycolic acid copolymer (MH-PLGA). To demonstrate the anti-inflammatory effects of the MH-PLGA scaffold, an animal model should be sensitive to inflammatory responses. The interleukin-10 knockout (IL-10 KO) mouse is a widely used bowel disease model for evaluating inflammatory responses, however, few studies have evaluated this mouse for the anti-inflammatory scaffold. To compare the sensitivity of the inflammatory reaction, the PLGA scaffold was implanted into IL-10 KO and C57BL/6 mouse kidneys. Morphology, histology, immunohistochemistry, and gene expression analyses were carried out at weeks 1, 4, 8, and 12. The anti-inflammatory effect and renal regeneration potency of the MH-PLGA scaffold was also compared to those of PLGA in IL-10 KO mice. The PLGA scaffold-implanted IL-10 KO mice showed kidneys relatively shrunken by fibrosis, significantly increased inflammatory cell infiltration, high levels of acidic debris residue, more frequent CD8-, C-reactive protein-, and ectodysplasin A-positive cells, and higher expression of pro-inflammatory and fibrotic factors compared to the control group. The MH-PLGA scaffold group showed lower expression of pro-inflammatory and fibrotic factors, low immune cell infiltration, and significantly higher expression of anti-inflammatory factors and renal differentiation related genes compared to the PLGA scaffold group. These results indicate that the MH-PLGA scaffold had anti-inflammatory effects and high renal regeneration potency. Therefore, IL-10 KO mice are a suitable animal model for in vivo validation of novel anti-inflammatory scaffolds.

Interleukin-10 treatment attenuates sinus node dysfunction caused by streptozotocin-induced hyperglycaemia in mice

Diabetes, characterized by hyperglycaemia, causes sinus node dysfunction (SND) in several rodent models. Interleukin (IL)-10, which is a potent anti-inflammatory cytokine, has been reported to decrease in obese and diabetic patients. We tested the hypothesis that administration of IL-10 inhibits the development of SND caused by hyperglycaemia in streptozotocin (STZ)-induced diabetic mice. Six-week old CL57/B6 (WT) mice were divided into the following groups: control, STZ injection, and STZ injection with systemic administration of IL-10. IL-10 knockout mice were similarly treated. STZ-induced hyperglycaemia for 8 weeks significantly depressed serum levels of IL-10, but increased several proinflammatory cytokines in WT mice. STZ-induced hyperglycaemia-reduced resting heart rate (HR), and attenuated HR response to isoproterenol in WT mice. In isolated perfused heart experiments, corrected-sinus node recovery time was prolonged in WT mice with STZ injection. Sinus node tissue isolated from the WT-STZ group showed fibrosis, abundant infiltration of macrophages, increased production of reactive oxygen species (ROS), and depressed hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4). However, the changes observed in the WT-STZ group were significantly attenuated by IL-10 administration and were further exaggerated in IL-10 knockout mice. In cultured cells, preincubation of IL-10 suppressed hyperglycaemia-induced apoptotic and profibrotic signals, and overproduction of ROS. IL-10 markedly inhibited the high glucose-induced p38 activation, and activated signal transducer and activator of transcription (STAT) 3 phosphorylation. Our results suggest that IL-10 attenuates ROS production, inflammation and fibrosis, and plays an important role in the inhibition of hyperglycaemia-induced SND by suppression of HCN4 downregulation. In addition, IL-10-mediated inhibition of p38 is dependent on STAT3 phosphorylation.

Different Characteristics of Hepcidin Expression in IL-6+/+ and IL-6-/- Neurons and Astrocytes Treated with Lipopolysaccharides.

A region-specific regulation of inflammation on the expression hepcidin in the brain has been demonstrated, however, it remains unknown whether there is also a cell-specific regulation of inflammation on hepcidin in the brain. Here, we investigated the effects of lipopolysaccharides (LPS) on the expression of hepcidin mRNA and also the expression of IL-6 mRNA, the phosphorylation of STAT3 and the expression of ferroportin 1 (Fpn1) and ferritin light chain (Ft-L) proteins in neurons and astrocytes obtained from wild type (IL-6+/+) and IL-6 knockout (IL-6-/-) mice. We demonstrated that the responses of the expression of hepcidin and IL-6 mRNAs, the phosphorylation of STAT3, and the expression of Fpn1 protein to LPS in IL-6+/+ astrocytes and also the responses of the expression of hepcidin mRNA, the phosphorylation of STAT3 and the expression of Fpn1 protein to IL-6 in IL-6-/- astrocytes were much stronger than those in IL-6+/+ and IL-6-/- neurons. A significant increase in Ft-L was found in LPS-treated IL-6+/+ and IL-6-treated IL-6-/- astrocytes, but not in LPS-treated IL-6+/+ and IL-6-treated IL-6-/- neurons. Our findings provide in vitro evidence for the existence of a cell-specific regulation of LPS on the expression of hepcidin and also Ft-L in the brain.

Worsened brain edma and the level of serum HMGB1 and S100B of IL-4 knockout mice after traumatic brain injury

Objective To investigate the changes of brain edma and expression of blood high mobility group box 1(HMGB1) and calcium binding protein S100B after traumatic brain injury (TBI) in IL-4 knockout (IL-4 KO) mice, and to explore the effects of IL-4 on traumatic brain injury. Methods Twenty male wild type (WT) or twenty male IL-4 KO BALB/cJ mice were randomly divided into WT sham TBI group, WT TBI group, IL-4 KO sham TBI group and IL-4 KO TBI group(n=10 in each group). The model of traumatic brain injury was established by the free falling body epidural impact method, then the brain water content was measured.The expression of aquaporin-4 (APQ4) and HMGB1 in injured brain of each group was detected by Western blot, and the concentration of HMGB1 and S100B in serum was detected by ELISA assay. Results (1) The brain water content of injured lateral brain of BALB/cJ mice with IL-4 gene knockout was significantly higher than that of wild type mice with brain injury model (WT group: (80.03±0.35)%; IL-4 KO group: (81.93±0.41)%; P<0.05). (2) The Western blot showed that the expression of AQP4 and HMGB1 in brain tissue of BALB/cJ mice with IL-4 gene knockout was significantly higher than those in wild type mice after traumatic brain injury.(3) The results of ELISA showed that the levels of HMGB1 and S100B in the serum of IL-4 knockout BALB/cJ mice were significantly higher than those of wild type mice (HMGB1: WT group: (9.21±0.74)ng/ml; IL-4 gene knock-out group: (13.39±1.33)ng/ml, P<0.05; S100B protein: WT group: (11.11±0.84)pg/ml; IL-4 KO group: (18.11±2.02)pg/ml, P<0.05 ). Conclusion The brain tissue water content and the expression of APQ4 are increased in IL-4 KO TBI mice.The expression of HMGB1 in brain issue and serum and S100B in serum are also up-regulated. Key words: Traumatic brain injury; Interleukin-4; Brain edema; High mobility group box 1; Calcium binding protein S100B; Mice

Interleukin 10 Treatment Ameliorates High-Fat Diet-Induced Inflammatory Atrial Remodeling and Fibrillation.

Obesity, characterized by systemic low-grade inflammation, is considered a well-known risk for atrial fibrillation. In fact, IL-10 (interleukin 10), which is a potent anti-inflammatory cytokine, has been reported to decrease in obese and diabetic patients. We tested the hypotheses forwarding that genetic deletion of IL-10 exacerbates high-fat diet (HFD)-induced obesity-caused atrial inflammation, lipidosis, fibrosis, and fibrillation and that IL-10 therapy inhibits this pathology. Eight- to 10-week-old male CL57/B6 (wild-type) mice and IL-10 knockout mice were divided into a 12-week HFD group and a 12-week normal-fat diet (NFD) group, respectively. In addition, the effect of IL-10 administration was also investigated. HFD-induced obesity for 12 weeks significantly depressed serum levels of IL-10 but were found to increase several proinflammatory cytokines in wild-type mice. Adverse atrial remodeling, including atrial inflammation, lipidosis, and fibrosis, was induced in both wild-type and IL-10 knockout mice by HFD. Vulnerability to atrial fibrillation was also significantly enhanced by HFD. With regard to epicardial and pericardial adipose tissue, the total amount of epicardial adipose tissue+pericardial adipose tissue volume was increased by HFD. Besides, proinflammatory and profibrotic cytokines of epicardial adipose tissue+pericardial adipose tissue were also upregulated. In contrast, the protein level of adiponectin was downregulated by HFD. These HFD-induced obesity-caused adverse effects were further exaggerated in IL-10 knockout mice in comparison to wild-type mice. Systemic IL-10 administration markedly ameliorated HFD-induced obesity-caused left atrial remodeling and vulnerability to atrial fibrillation, in addition to improving the quality of epicardial adipose tissue+pericardial adipose tissue. Our results highlight IL-10 treatment as a potential therapeutic approach to limit the progression of HFD-induced obesity-caused atrial fibrillation.

Non-coding RNA regulation of immunity

Abstract Non-coding RNAs (ncRNAs) comprise a diverse range of biologically functional RNA species that include long ncRNAs (lncRNA) and short ncRNAs such as piwi-interacting RNAs (piRNA) and microRNAs (miRNA). These ncRNAs are capable of regulating gene expression via distinct mechanisms depending on their structure---miRNAs bind specific messenger RNAs (mRNA) to block translation while lncRNAs tend to bind chromatin to modify chromatin states to interfere with gene expression. NcRNAs have been identified in numerous diseases as having altered expression. To further elucidate the role of ncRNAs in immunity, we conducted a screen of lncRNA expression in the interleukin-10 knockout mouse (IL-10−/−) model of intestinal inflammation. This screen identified several candidate lncRNAs with dysregulated expression. Importantly, our data demonstrate that Malat1 and Hoxa11as expression was significantly decreased in mice with severe intestinal pathology. These results suggest that an association exists between these lncRNAs and the progression of disease in the IL-10−/− mouse model of colitis.

Random mutagenesis unveils novel host-pathogen interactions during colonic bacterial infections in immunocompromised hosts

Abstract Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and the rodent equivalent Citrobacter rodentium (CR) are important causative agents of foodborne diseases. A/E pathogen infections cause severe morbidity and mortality in immunocompromised hosts with low interleukin-22 (IL-22); however, the crucial host-pathogen interactions and the pivotal A/E virulence proteins (effectors) under immunocompromised conditions, remain elusive. We conducted a functional screening of a CR mutant library consisting of ~2,000 mutant strains, generated via transposon-mediated random mutagenesis, and identified several avirulent strains, which abolish CR-induced severe morbidity and mortality in IL-22 knockout (Il22−/−) mice. Recapitulating the attenuated virulence phenotype by the transposon-interrupted mutant strains, genomic deletion of each identified effector in CR dramatically attenuates CR-caused lethality in Il22−/− mice. Interestingly, genetic deletion of each identified effector gene in CR genome exhibits no impact on the colonization and proliferation of CR in the infected animals; however, the deficiencies in these effectors substantially impede the colonic inflammatory response in the CR-infected Il22−/− mice, through distinct cellular and molecular mechanisms. Hence, our findings unveil a set of novel A/E pathogen effectors pathologically relevant to the severe morbidity and mortality in Il22−/− animals, which could provide new strategies to control A/E pathogen infections under the low IL-22 immunocompromised conditions, which are frequently associated with chronic HIV infection, organ transplantation, and other diseases.

Interleukin 6 Knockout Inhibits Aging-Related Accumulation of p53 in the Mouse Myocardium.

Interleukin 6 (IL6) and p53 are linked by mutual regulatory mechanisms and are both upregulated in aging. The aim of this study was to evaluate the effects of aging and IL6 on expression of p53 in the mouse heart. Male C57BL6/J wild-type and IL6 knockout mice at the age of 4-5 months (young adult) and 24-30 months (old) were used. Myocardial expression of proteins such as p53, p21, Mdm2, and phospho-Akt/Akt was estimated using Western blotting and expression of p53 and p21 mRNA using real-time polymerase chain reaction. Expression of p53 protein was lower in IL6 knockout hearts than in wild-type hearts. Aging caused significant upregulation of p53 protein level; however, it was significantly higher in old wild-type hearts than in old IL6 knockout hearts (p < .05). Similar p53 mRNA levels in all groups implied IL6 influence on age-related proteasomal degradation of p53. Localization of p53 mainly in the extranuclear compartment and lack of p21 upregulation in aged hearts may suggest quenched transcriptional activity of p53 despite increased abundance of p53. We conclude that lack of IL6 attenuates expression of p53 protein in the hearts of young mice and diminishes its accumulation with aging by post-transcriptional mechanisms; however, this is not related to altered phenotype of aging heart.

IL-17 deficiency attenuates acetaminophen-induced hepatotoxicity in mice

Acetaminophen (APAP) overdose results in the production of reactive oxygen species (ROS), hepatocyte necrosis, and cell death, and leads to acute liver failure. Interleukin-17 (IL-17), a pro-inflammatory cytokine, plays a key role in the recruitment of neutrophils into sites of inflammation and subsequent damage after liver ischemia-reperfusion injury. In this study, we employed IL-17 knockout (KO) mice to investigate the role of IL-17 in APAP-induced hepatotoxicity. IL-17 wide type (WT) and IL-17 KO mice received an intraperitoneal injection of APAP (300 mg/kg). After 16 h of treatment, the hepatic injury, inflammatory mediators, immune cell infiltration, and western blotting were examined and analyzed. The serum alanine transferase (ALT) enzyme levels and hepatic myeloperoxidase (MPO) activity were significantly elevated 16 h after APAP treatment in the WT mice. IL-17 deficiency significantly attenuates APAP-induced liver injury, MPO activity, pro-inflammatory cytokines (tumor necrosis factor-α, IL-6 and interferon-γ) levels and inflammatory cell (neutrophils, macrophage) infiltration in the liver. Moreover, phosphorylated extracellular signal-regulated kinase (ERK) was significantly decreased at 16 h after APAP treatment in the IL-17 KO mice compared with the IL-17 WT mice. Our data suggests that IL-17 plays a pivotal role in APAP-induced hepatotoxicity through modulation of inflammatory response, and perhaps in part through the ERK signaling pathway. Blockade of IL-17 could be a potential therapeutic target for APAP-induced hepatotoxicity.

IL-6 knockout mice are protected from cocaine-induced kindling behaviors; possible involvement of JAK2/STAT3 and PACAP signalings

IL-6 has been recognized as an anticonvulsant against certain neuroexcitotoxicities. We aimed to investigate on the interactive role between IL-6 and PACAP in cocaine-induced kindling behaviors. Although we found that cocaine (45 mg/kg, i.p./day x 5) significantly increased IL-6 and TNF-α expression, it resulted in a decrease in IFN-γ expression. We observed that the cocaine-induced increase in IL-6 expression was more pronounced than that in TNF-α expression. Genetic depletion of IL-6 significantly activated cocaine kindling behaviors. This phenomenon was also consistently observed in WT mice that received a neutralizing IL-6 receptor antibody. Cocaine-treated IL-6 knockout mice exhibited significantly decreased PACAP and PACAP receptor (PAC1R) mRNA levels and significantly increased TNF-α gene expression. TNF-α knockout mice were protected from cocaine kindling via an up-regulation of IL-6, phospho-JAK2/STAT3, PACAP, and PAC1R levels, which produced anti-apoptotic effects. Recombinant IL-6 protein (rIL-6, 2 μg, i.v./mouse/day x 5) also up-regulated phospho-JAK2/STAT3, PACAP, and PAC1R mRNA levels, leading to anti-apoptotic effects in IL-6 knockout mice. Consistently, AG490, a JAK2/STAT3 inhibitor, and PACAP 6–38, a PAC1 receptor antagonist, counteracted rIL-6-mediated protection. Combined, our results suggest that IL-6 gene requires up-regulation of phospho-JAK2/STAT3, PACAP, and PAC1R and down-regulation of the TNF-α gene to modulate its anticonvulsive/neuroprotective potential.

MP23-03 IL-6/STAT3 SIGNALING IS A HOST DETERMINANT OF UPEC VIRULENCE VIA ALTERED BACTERIAL INVASION AND INTRACELLULAR BACTERIAL COMMUNITY FORMATION

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder II1 Apr 2018MP23-03 IL-6/STAT3 SIGNALING IS A HOST DETERMINANT OF UPEC VIRULENCE VIA ALTERED BACTERIAL INVASION AND INTRACELLULAR BACTERIAL COMMUNITY FORMATION Christina Ching, Sudipti Gupta, and Brian Becknell Christina ChingChristina Ching More articles by this author , Sudipti GuptaSudipti Gupta More articles by this author , and Brian BecknellBrian Becknell More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.735AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our understanding of bacterial virulence in urinary tract infection (UTI) is primarily limited to identification of bacteria-specific determinants. Host mediators are less well known. IL-6 is a cytokine that is induced during UTI and results in subsequent Stat3 phosphorylation, but without readily apparent functional significance. We sought to evaluate the impact of IL-6/Stat3 signaling on host susceptibility to UTI via urothelial bacterial invasion and intracellular bacterial community formation. METHODS We performed in vivo and in vitro studies in order to evaluate the impact of IL-6 and Stat3 in bacterial invasion. We infected 6 week old female wild type (WT) C57BL/6J, IL-6 knock out (KO), or tamoxifen-inducible Stat3 conditional KO mice transurethrally with UPEC. We evaluated bladder and urine bacterial burden at varying time points of sacrifice by serial plating of tissue homogenates on LB agar and IBC formation via beta-galactosidase staining. We similarly evaluated the impact of recombinant IL-6 rescue in IL-6 KO mice on IBC formation and antimicrobial formation. We evaluated the impact of recombinant IL-6 incubation and Stat3 inhibition on intracellular bacterial invasion with gentamicin protection assay in serum starved 5637 urothelial cells. Results were evaluated by Mann-Whitney U test with p <0.05 being significant. RESULTS We observed an early transient (6 hours post inoculation - hpi) increase in bladder bacterial burden in our IL-6 KO mice compared to the WT controls, with a subsequent and persistent increase in urine bacterial burden starting at 24 hpi. This matched an initial significant difference in bladder IBC number in the IL-6 KO vs. WT that resolved by 24 hpi and was confirmed in our Stat3 CKO mice. Recombinant intravesical IL-6 rescued this phenotype in the IL-6 KO mouse and also significantly increased antimicrobial peptide expression. Similarly, we found that there was a significant decrease in bacterial invasion in vitro when 5637 cells were preincubated with IL-6. Stat3 inhibition resulted in a reversal of this phenotype with an increase in bacterial invasion on par with if not above unstimulated cells. CONCLUSIONS IL-6 plays an important role in host susceptibility to UTI as a mediator of bacterial invasion and IBC formation, potentially via antimicrobial peptide formation. Given IBC formation represents a significant bottleneck in UPEC survival during cystitis, these findings could suggest a role of IL-6 in UTI persistence and chronicity. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e282 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Christina Ching More articles by this author Sudipti Gupta More articles by this author Brian Becknell More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Deletion of protein kinase B2 preserves cardiac function by blocking interleukin-6-mediated injury and restores blood pressure during angiotensin II/high-salt-diet-induced hypertension.

Protein kinase B2 (AKT2) is implicated in cardiomyocyte survival during various stress conditions. However, the role of AKT2 in heart function, cardiac hypertrophy and blood pressure (BP) control during hypertension is not fully understood. Therefore, we sought to determine whether the deletion of AKT2 protects cardiac function during angiotensin II/high-salt-diet (AngII/HSD) treatment and find out the signaling pathway. Male C57BL/6J (wild type), AKT2 knockout and interleukin (IL)-6 knockout mice were fed a 4% NaCl diet for 5 weeks. In the last week, mice were split in two groups and infused subcutaneously with either vehicle or AngII (1.5 μg/h per mouse) for 1 week. Then, BP and cardiac function were assessed. Immunohistology of IL-6 and monocyte chemoattractant protein 1 was performed to detect inflammation in the heart. Masson's trichrome staining was performed to evaluate cardiac fibrosis. Heart tissue homogenates and neonatal mice cardiomyocytes were collected to analyze oxidative stress. Compared with wild-type mice, AKT2 knockout mice maintained BP and showed better left ventricle ejection fraction, lower level of fibrosis, reduced oxidative stress, reduced IL-6 expression and less macrophage infiltration, when treated with AngII/HSD. IL-6 knockout mice treated with AngII/HSD also showed alleviated left ventricular function, fibrosis, oxidative stress and macrophage infiltration compared with wild type. AKT2 deficiency prevents the development of AngII/HSD-induced hypertension, cardiac dysfunction and myocardial injury including oxidative stress, fibrosis and inflammation by suppressing IL-6 expression. These data reveal an important role of the AKT2-IL-6 pathway in mediating AngII/HSD-induced hypertension and cardiomyopathy.

Alterations in Docosahexaenoic Acid-Related Lipid Cascades in Inflammatory Bowel Disease Model Mice.

Inflammatory bowel disease (IBD) is an intestinal disorder, involving chronic and relapsing inflammation of the digestive tract. Dysregulation of the immune system based on genetic, environmental, and other factors seems to be involved in the onset of IBD, but its exact pathogenesis remains unclear. Therefore, radical treatments for ulcerative colitis and Crohn's disease remain to be found, and IBD is considered to be a refractory disease. The aim of this study is to obtain novel insights into IBD via metabolite profiling of interleukin (IL)-10 knockout mice (an IBD animal model that exhibits a dysregulated immune system). In this study, the metabolites in the large intestine and plasma of IL-10 knockout mice were analyzed. In our analytical system, two kinds of analysis (gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry) were used to detect a broader range of metabolites, including both hydrophilic and hydrophobic metabolites. In addition, an analysis of lipid mediators in the large intestine and ascites of IL-10 knockout mice was carried out. The levels of a variety of metabolites, including lipid mediators, were altered in IL-10 knockout mice. For example, high large intestinal and plasma levels of docosahexaenoic acid (DHA) were observed. In addition, arachidonic acid- and DHA-related lipid cascades were upregulated in the ascites of the IL-10 knockout mice. Our findings based on metabolite profiles including lipid mediators must contribute to development of researches about IBD.

Impaired Inferior Vena Cava Relaxation but not Contraction in Two Models of Murine Acute Deep Venous Thrombosis

Venous thrombosis (VT) damages the vein wall, both physically with prolonged distention and from inflammation. These factors underlie post-thrombotic syndrome (PTS). Interleukin 6 (IL-6) may play a role in experimental PTS and vein wall responses. Prior assessment of post-thrombotic vein wall injury has used static measures such as histology and immunologic assays. The purpose of this study was to use myography to quantify the changes in contraction and relaxation of murine vessels exposed to an acute thrombus. Wild-type (WT) C57BL/6 mice were used to determine baseline vein wall passive tension on a DMT 610M myograph (DMT, Ann Arbor, Mich), including dosing concentrations of phenylephrine (Phe) and acetylcholine (Ach). WT and IL-6 knockout mice underwent VT using inferior vena cava (IVC) ligation (complete stasis) and stenosis (partial stasis), and no-surgery mice were used as controls. Mice were harvested at 2 days (2D) and analyzed using a myograph. Vessels were stimulated with Phe and Ach to stimulate a contraction and relaxation response. Immunohistochemistry for CD31 was used to identify endothelial cells on WT no-surgery and WT 2D ligation vessels. Optimal passive tension was determined to be 2 mN, with the optimal concentrations of Phe and Ach being 7E-3M and 1E-5M, respectively. There was no significant difference in contraction on exposure to Phe between the WT no-thrombus mice, WT 2D ligation, and WT 2D stenosis mice and the IL-6 knockout mice with or without thrombus (all P > .05). When mice were treated with Ach, there was significantly more relaxation in the nonthrombosed control IVC segments than in those mice IVC segments that had a 2D thrombus of either ligation- or stenosis-derived mechanisms (Fig). The CD31 staining confirmed the presence of endothelium in the WT 2D IVC thrombus mice, ruling out tissue lost during harvesting. Compared with controls, acute thrombosis with total or partial stasis did not impair IVC contractile responses, suggesting no effect on medial vascular smooth muscle response. The relaxation response, however, was significantly reduced in the post-thrombotic groups. These findings suggest that at acute time points, VT impairs the endothelial function of a vein wall while retaining the vascular smooth muscle cell function and may be a mechanism that promotes PTS.

Interleukin-6/Stat3 signaling has an essential role in the host antimicrobial response to urinary tract infection

The signaling networks regulating antimicrobial activity during urinary tract infection (UTI) are incompletely understood. Interleukin-6 (IL-6) levels increase with UTI severity, but the specific contributions of IL-6 to host immunity against bacterial uropathogens are unknown. To clarify this we tested whether IL-6 activates the Stat3 transcription factor, to drive a program of antimicrobial peptide gene expression in infected urothelium during UTI. Transurethral inoculation of uropathogenic Escherichia coli led to IL-6 secretion, urothelial Stat3 phosphorylation, and activation of antimicrobial peptide transcription, in a Toll-like receptor 4-dependent manner in a murine model of cystitis. Recombinant IL-6 elicited Stat3 phosphorylation in primary urothelial cells invitro, and systemic IL-6 administration promoted urothelial Stat3 phosphorylation and antimicrobial peptide expression invivo. IL-6 deficiency led to decreased urothelial Stat3 phosphorylation and antimicrobial peptide mRNA expression following UTI, a finding mirrored by conditional Stat3 deletion. Deficiency in IL-6 or Stat3 was associated with increased formation of intracellular bacterial communities, and exogenous IL-6 reversed this phenotype in IL-6 knockout mice. Moreover, chronic IL-6 depletion led to increased renal bacterial burden and severe pyelonephritis in C3H/HeOuJ mice. Thus, IL-6/Stat3 signaling drives a transcriptional program of antimicrobial gene expression in infected urothelium, with key roles in limiting epithelial invasion and ascending infection.

Chemotherapy-induced pain is promoted by enhanced spinal adenosine kinase levels through astrocyte-dependent mechanisms.

Development of chemotherapy-induced neuropathic pain (CINP) compromises the use of chemotherapy and greatly impacts thousands of lives. Unfortunately, there are no Food and Drug Administration-approved drugs to prevent or treat CINP. Neuropathological changes within CNS, including neuroinflammation and increased neuronal excitability, are driven by alterations in neuro-glia communication; but, the molecular signaling pathways remain largely unexplored. Adenosine is a potent neuroprotective purine nucleoside released to counteract the consequences of these neuropathological changes. Adenosine signaling at its adenosine receptors (ARs) is dictated by adenosine kinase (ADK) in astrocytes, which provides a cellular sink for the removal of extracellular adenosine. We now demonstrate that chemotherapy (oxaliplatin) in rodents caused ADK overexpression in reactive astrocytes and reduced adenosine signaling at the A3AR subtype (A3AR) within the spinal cord. Dysregulation of ADK and A3AR signaling was associated with increased proinflammatory and neuroexcitatory interleukin-1β expression and activation of nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome, but not putative oxaliplatin-associated GSK3β transcriptional regulation. Intrathecal administration of the highly selective A3AR agonist MRS5698 attenuated IL-1β production and increased the expression of potent anti-inflammatory and neuroprotective IL-10. The effects of MRS5698 were blocked by attenuating IL-10 signaling in rats with intrathecal neutralizing IL-10 antibody and in IL-10 knockout mice. These findings provide new molecular insights implicating astrocyte-based ADK-adenosine axis and nucleotide-binding oligomerization domain-like receptor protein 3 in the development of CINP and IL-10 in the mechanism of action of A3AR agonists. These findings strengthen the pharmacological rationale for clinical evaluation of A3AR agonists already in advanced clinical trials as anticancer agents as an adjunct to chemotherapy.