Metabolic danger signals, uric acid and ATP, mediate inflammatory cross-talk between hepatocytes and immune cells in alcoholic liver disease
|
|
- Stephanie Burns
- 5 years ago
- Views:
Transcription
1 Brief Conclusive Report Metabolic danger signals, uric acid and ATP, mediate inflammatory cross-talk between hepatocytes and immune cells in alcoholic liver disease Jan Petrasek, Arvin Iracheta-Vellve, Banishree Saha, Abhishek Satishchandran, Karen Kodys, Katherine A. Fitzgerald, Evelyn A. Kurt-Jones, and Gyongyi Szabo 1 Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA RECEIVED DECEMBER 5, 2014; REVISED MARCH 27, 2015; ACCEPTED APRIL 14, DOI: /jlb.3AB R ABSTRACT Inflammation defines the progression of ALD from reversible to advanced stages. Translocation of bacterial LPS to the liver from the gut is necessary for alcoholinduced liver inflammation. However, it is not known whether endogenous, metabolic danger signals are required for inflammation in ALD. Uric acid and ATP, 2 major proinflammatory danger signals, were evaluated in the serum of human volunteers exposed to a single dose of ethanol or in supernatants of primary human hepatocytes exposed to ethanol. In vitro studies were used to evaluate the role of uric acid and ATP in inflammatory cross-talk between hepatocytes and immune cells. The significance of signaling downstream of uric acid and ATP in the liver was evaluated in NLRP3- deficient mice fed a Lieber-DeCarli ethanol diet. Exposure of healthy human volunteers to a single dose of ethanol resulted in increased serum levels of uric acid and ATP. In vitro, we identified hepatocytes as a significant source of these endogenous inflammatory signals. Uric acid and ATP mediated a paracrine inflammatory cross-talk between damaged hepatocytes and immune cells and significantly increased the expression of LPS-inducible cytokines, IL-1b and TNF-a, by immune cells. Deficiency of NLRP3, a ligand-sensing component of the inflammasome recognizing uric acid and ATP, prevented the development of alcoholinduced liver inflammation in mice and significantly ameliorated liver damage and steatosis. Endogenous metabolic danger signals, uric acid, and ATP are involved in inflammatory cross-talk between hepatocytes and immune cells and play a crucial role in alcoholinduced liver inflammation. J. Leukoc. Biol. 98: ; Abbreviations: AIM2 = absent in melanoma 2, ALD = alcoholic liver disease, ASC = apoptosis-associated speck-like protein containing a caspaserecruitment domain, HS = heat shock, LDH = lactate dehydrogenase, LMNC = liver mononuclear cell, NLRP3 = nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3, UMASS = University of Massachusetts, WT = wild-type, z-vad-fmk, = z-val-ala-asp-fluoromethylketone Introduction Progression of ALD to advanced form is tightly linked to liver inflammation (reviewed in ref. [1]). We have previously demonstrated that the proinflammatory cytokine, IL-1b is required for the development of alcohol-induced liver inflammation, steatosis, and injury and that the pathogenic effect of IL- 1b is specific to resident liver macrophages (Kupffer cells) [2]. We also demonstrated that in the mouse model of ALD, the conversion of inactive pro-il-1b to active IL-1b required inflammasome, an intracellular multicomponent complex that triggers inflammation in response to endogenous danger signals. Synthesis of active IL-1b is a 2-step process. In the 1st step, microbial-derived signals up-regulate pro-il-1b, resulting in synthesis of inactive pro-il-1b. This step is triggered by binding of bacterial LPS, derived from the gut, to the TLR4 on Kupffer cells and provides an initial proinflammatory signal in ALD [3 7]. Deficiency in TLR4 prevents up-regulation of pro-il-1b in the liver of alcohol-fed mice [8]. In the 2nd step, signals derived from host cells ( danger signals) activate the inflammasome, resulting in cleavage of pro-il- 1b into the active IL-1b [9]. The nature of endogenous danger signals that activate inflammasome and IL-1b in ALD is not known. Here, we show that hepatocytes damaged by ethanol release endogenous metabolic danger molecules, uric acid, and ATP, which are recognized by liver immune cells as inflammatory signals. Uric acid and ATP from damaged hepatocytes are required for processing and activation of IL-1b, a cytokine requiring inflammasome activation that is crucial in the pathogenesis of ALD [2]. Our data suggest that the release of these molecules represents signals permissive for the proinflammatory effect of LPS in ALD. MATERIALS AND METHODS Animal studies Six- to 8-week-old female C57Bl/6 WT (The Jackson Laboratory, Bar Harbor, ME, USA), NLRP3-deficient mice (kind gift of Dr. Amy Hise, Case Western 1. Correspondence: Dept. of Medicine, University of Massachusetts Medical School, LRB 215, 364 Plantation St., Worcester, MA 01605, USA. gyongyi.szabo@umassmed.edu /15/ Society for Leukocyte Biology Volume 98, August 2015 Journal of Leukocyte Biology 249
2 Reserve University, Cleveland, OH, USA), all on a C57Bl/6 background, were used. Some animals were fed with the Lieber-DeCarli ad libitum diet (Bioserv, Frenchtown, NJ, USA) with 5% vol/vol ethanol (36%-derived calories); pairfed control mice matched the alcohol-derived calories with dextran-maltose. Specifically, mice received a fresh Lieber-DeCarli diet in 50 ml feeders daily, between 7:00 PM and 8:00 PM. At the conclusion of the experiment, we collected blood and harvested livers between 8:00 AM and 9:00 AM [2]. Serum ethanol levels did not significantly differ between the genotypes. All animals received proper care in agreement with animal protocols approved by the Institutional Animal Care and Use Committee of the UMASS Medical School (Worcester, MA, USA). Administration of ethanol to human volunteers Peripheral blood was obtained by venipuncture with heparin anticoagulation (0.5 mg/ml blood) from 11 healthy volunteers (aged years; 9 women, 2 men) from the clerical and professional staff at UMASS Medical Center. Volunteers abstained from alcoholic beverages for at least 48 hours before blood donation. The average alcohol use was,6 drinks/week for women and,9 drinks/week for men. Alcohol-use habits of blood donors were determined by a health assessment questionnaire that incorporates the AUDIT and CAGE tests and Health Screening Survey to identify frequency and quantities of alcohol use. In brief, blood samples ( ml) were obtained on day 1 before alcohol consumption, as well as at indicated timepoints after the ethanol uptake. Alcohol was given as vodka, 2 ml/kg body weight (0.85 g ethanol/kg body weight), diluted with orange juice, for a total volume of 300 ml. The alcohol was consumed within 30 minutes. Three male volunteers from the professional staff at UMASS Medical Center that were not exposed to ethanol were used as controls. The study was reviewed and approved by the Institutional Committee for Protection of Human Subjects in Research. Isolation of primary mouse hepatocytes and LMNCs Anesthetized animals were perfused by way of portal vein with saline solution, followed by enzymatic digestion, as described in refs. [2, 10]. The hepatocytes were separated by centrifugation, and LMNCs were purified by centrifugation in Percoll gradient. LPS was purchased from Sigma (St. Louis, MO, USA), and IL-1b-specific neutralizing antibody (AF-401) was purchased from R&D Systems (Minneapolis, MN, USA) and used according to the manufacturer s specifications. Primary human hepatocytes Primary human hepatocytes were obtained from the NIH Liver Tissue Cell Distribution System (Minneapolis, MN, USA; Pittsburgh, PA, USA; and Richmond, VA, USA), which was funded by NIH Contract #N01-DK-7-004/ HHSN C. Human hepatocytes were cultured in low-glucose DMEM, supplemented with 10% FBS and 1% insulin, transferrin, and selenium solution (Gibco Life Technologies, Grand Island, NY, USA). Suramin was purchased from Sigma, z-vad-fmk and ATP were from InvivoGen (San Diego, CA, USA), cytochalasin-d was from EMD-Calbiochem (La Jolla, CA, USA), and Ca-074-Me was from Invitrogen (Grand Island, NY, USA). Isolation of human PBMCs Human PBMCs were separated from blood of healthy volunteers by centrifugation in a Ficoll gradient as described in ref. [11]. Biochemical assays Serum ALT was determined by use of a kinetic method (D-Tek, Bensalem, PA, USA). Colorimetric assays were used to measure serum uric acid (Abcam, Cambridge, MA, USA), liver triglycerides (Wako Chemicals, Richmond, VA, USA), and LDH activity in cell-culture supernatants (Abcam). Chemiluminiscent assay was used to measure ATP in the serum or in cell-culture supernatants (CellTiter-Glo; Promega, Madison, WI, USA). Cytokine measurement TNF-a was measured by use of specific anti-mouse ELISA from BioLegend (San Diego, CA, USA). IL-1b was measured by use of specific anti-mouse ELISA (R&D Systems) that recognizes pro-il-1b and cleaved IL-1b. Protein quantification Liver whole-cell lysates were extracted as described previously [2, 12]. Equal amounts of proteins were separated on a polyacrylamide gel and transferred to a nitrocellulose membrane. Target proteins were detected by Western blot and immunostaining with specific primary antibody, followed by HRP-labeled secondary antibody. The specific immunoreactive bands of interest were detected by chemiluminescence (Amersham, Piscataway, NJ, USA). Digital system (ImageQuant LAS 4000; GE Healthcare, Uppsala, Sweden) was used for image acquisition. Blots labeled as short exposure and long exposure (see Fig. 5) were exposed for 1 or 10 minutes, respectively. Antibodies specific for total caspase-1 and the cleaved p10 fragment of caspase-1 were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Antibody specific for total and cleaved IL-1b was from R&D Systems, and antibody recognizing the total and cleaved forms of caspase-3 was from Cell Signaling Technology (Danvers, MA, USA). b-tubulin antibodies were from Abcam. Histopathological analysis Liver sections were stained with H&E or Oil Red O by use of standard protocols analyzed by microscopy. Statistical analysis Statistical significance was determined by use of 2-sided t-test; ANOVA and Dunnett s multiple comparison post-test were used to compare the means of multiple groups. Two-way ANOVA was used (see Fig. 5D) to determine the global effect of genotype or treatment on serum ALT. Unless stated otherwise, data are shown as means 6 SEM and were considered statistically significant at P, We used SPSS 19.0 (IBM SPSS, Chicago, IL, USA) to perform statistical analyses. RESULTS AND DISCUSSION Endogenous inflammasome activators, ATP, and uric acid are increased in humans after acute alcohol bingedrinking Liver inflammation induced by alcohol is dependent on activation of the inflammasome and IL-1b [2], implying involvement of endogenous danger signals in triggering liver inflammation in ALD. Given the effect of ethanol on mitochondrial function and metabolism of purine nucleotides, we studied the role of ATP and uric acid, 2 well-characterized metabolic danger signals and activators of inflammasome [9]. First, we asked whether alcohol modulates levels of uric acid and ATP. We evaluated uric acid and ATP in the serum of healthy human volunteers after a single binge-drinking of 0.8 mg/kg ethanol. We observed an early and statistically significant increase in serum levels of uric acid (Fig. 1A) and ATP (Fig. 1B) in alcohol-exposed volunteers but not in individuals not exposed to ethanol. In further experiments, we observed that treatment of primary human hepatocytes with ethanol resulted in hepatocyte death and in significantly increased levels of uric acid and ATP in the cell-culture supernatants (Fig. 2A). Similar results were obtained when primary human hepatocytes were 250 Journal of Leukocyte Biology Volume 98, August
3 Petrasek et al. Hepatocyte-derived uric acid and ATP in liver inflammation used HS as a strong stimulus inducing hepatocyte death. To evaluate whether uric acid and ATP are involved, we used uricase and apyrase to deplete uric acid and ATP, respectively, in the medium. We observed that stimulation of PBMCs with LPS, which provides only the 1st signal that induces pro-il-1b mrna, resulted in limited release of IL-1b proteininthesupernatant (Fig. 3A, group 3, indicated on x axis). Importantly, the release of IL-1b protein from PBMCs was significantly augmented by addition of supernatants from HS-damaged hepatocytes (Fig. 3A, group 4). This increase was partially abrogated by apyrase or uricase and completely abrogated by a combination of both. These data indicated that uric acid andatp,releasedfromdamagedhepatocytes,actasa2nd signal for inflammasome activation/il-1b maturation in PBMCs. Consistent with the mechanisms by which ATP or uric acid activate inflammasome, the release of IL-1b triggered by supernatants from HS hepatocytes required active inflammasome (inhibited by z-vad-fmk, groups 29 32), purinergic signaling (inhibited by suramin in Fig. 3A, groups 25 28), or cathepsin B-dependent signaling downstream of endosomes (inhibited by CA-074-Me in Fig. 3A, groups 21 24), respectively. However, induction of IL-1b seemed to be independent of endocytosis because of cytochalasin-d, an inhibitor of endocytosis (Fig. 3A, groups 17 20). Figure 1. Acute exposure to ethanol increases serum levels of uric acid and ATP in healthy human volunteers. Peripheral blood was obtained from 11 healthy volunteers, who consumed a single dose of ethanol, or from 3 volunteers not exposed to ethanol, as specified in the Materials andmethodssection.serumlevelsofuricacidandatpwere measured at indicated time-points. Data are presented as individual data points and as summary statistics (means 6 SEM). *P, 0.05 versus baseline. exposed to heat shock (HS) (Fig. 2B). These data indicated that alcohol-exposed hepatocytes release significant amounts uric acid and ATP, and this observation was consistent with previous reports that uric acid and ATP are present in high concentrations in hepatocytes [13, 14]. ATP and soluble uric acid released from damaged primary hepatocytes trigger the release of inflammasome-dependent cytokine IL-1b from immune cells IL-1b maturation is a 2-step process. LPS induces the pro-il-1b gene expression, but an additional, inflammasome-mediated signal is required for cleavage of pro-il-1b protein to mature IL-1b that is released from inflammatory cells [15]. To evaluate whether components released from damaged human hepatocytes serve as a 2nd signal for IL-1b release, we tested primary human hepatocytes and PBMCs. First, we asked whether damaged hepatocytes trigger the release of IL-1b from PBMCs. To increase the sensitivity of our experiments, we Figure 2. Damaged human hepatocytes release endogenous danger signals, uric acid, and ATP. Primary murine or human hepatocytes were treated for 3 hours with 800 mm ethanol, and levels of LDH, indicating hepatocyte death; ATP; and uric acid were evaluated in supernatants at indicated time-points (A). Primary murine or human hepatocytes were cultured for 30 minutes at 45 C (HS) or 37 C (control), and levels of LDH, ATP, and uric acid were evaluated in supernatants at indicated time-points (B). Stimulations were performed in triplicates. *P, 0.05 versus baseline. Volume 98, August 2015 Journal of Leukocyte Biology 251
4 CA-074-Me was closely associated with decreased secretion of TNF-a (Fig. 3B). This was consistent with the role of IL-1b in triggering the release of TNF-a via IL-1R signaling (refs. [16 18], and see below). Figure 3. ATP and soluble uric acid derived from damaged primary human hepatocytes trigger release of IL-1b and TNF-a from immune cells. Primary human hepatocytes seeded on 6-well plates were incubated at 37 C or 45 C (HS). Supernatant was collected at 8 hours. In the meantime, primary human PBMCs were isolated from 3 healthy donors, seeded on 96-well plates and incubated with or without LPS (100 ng/ml) for 2 hours. Meanwhile, supernatant harvested from human hepatocytes was preincubated with control media, uricase (10 U/ml), and/or apyrase (1 U/ml) for 1 hour at 37 C. PBMCs were incubated with cytochalasin-d (10 mg/ml), CA-074-Me (10 mm), suramin (0.1 mm), or z-vad-fmk (10 mm) for 1 hour. Three-fourths of PBMC media was then replaced with appropriate hepatocyte supernatant. After 8 hours, PBMC supernatant was collected, and IL-1b (A) and TNF-a (B) were measured. Stimulations were performed in triplicates. *P, 0.05 versus baseline; N.S., not significant. We also observed that the increased levels of IL-1b resulted in significantly enhanced levels of TNF-a and that inhibition of IL-1b releasebyuricase,apyrase,z-vad-fmk,suramin,or ATP and soluble uric acid released from alcoholexposed primary hepatocytes trigger the release of inflammasome-dependent cytokine IL-1b from liver immune cells In the next set of experiments, we asked whether uric acid and ATP released from damaged hepatocytes would activate liver immune cells (LMNCs). To answer that question, we used primary hepatocytes and LMNCs isolated from WT mice. In this scenario, we used ethanol to induce hepatocyte damage. Similar to experiment with human hepatocytes and PBMCs, we used uricase and apyrase to deplete uric acid or ATP, respectively. We observed that stimulation of LMNCs with LPS resulted in limited release of IL-1b, which was significantly augmented by addition of supernatants from alcoholdamaged hepatocytes (Fig. 4A). This increase was partially abrogated by apyrase or uricase and completely abrogated by a combination of both, supporting our finding of involvement of uric acid and ATP as the 2nd signal for inflammasome activation/il-1b maturation. The increased levels of IL-1b were associated with significantly enhanced levels of TNF-a (Fig. 4A, right). Next, we asked whether the effect of hepatocyte-derived danger signals on LMNCs is dose dependent. To increase the sensitivity of the in vitro system, we used HS to induce the sufficient extent of hepatocyte death. With the use of increasing concentrations of conditioned hepatocyte media, we observed increasing secretion of IL-1b by LPS-primed LMNCs. This effect was abrogated completely by adding uricase or apyrase (Fig. 4B). Taken together, these data indicated that the effect of hepatocyte-derived uric acid and ATP on inflammasome activation/il-1b release from LMNCs was dose dependent (Fig. 4B). Finally, we used murine LMNCs to investigate the role of IL-1b signaling in induction of TNF-a. Stimulation of LMNCs with LPS resulted in a significant, albeit minor, increase in IL-1b in the cell-culture supernatant (Fig. 4C, left). A further significant increase was achievedwithadditionofatpas a 2nd signal to LMNCs. This increase was abrogated by neutralization of IL-1b by use of a specific antibody, reflecting the dependency of IL-1b induction on an autocrine loop mediated by an IL-1R [17 19]. Along with increased levels of IL-1b, addition of ATP to LPS-stimulated LMNCs significantly increased levels of TNF-a in the supernatant (2.8-fold; Fig. 4C, right). This increase was attributable to increased levels of IL-1b, as stimulation of LMNCs in the presence of neutralizing antibodies against IL-1b did not result in any increase of TNF-a compared with baseline conditions (Fig.4C,right).Thesefindings were consistent with the previously reported role of IL-1b in triggering the release of TNF-a via IL-1R-dependent signaling [16 18], and extended the role of IL-1b in amplification of TNF-a expression to LMNCs. Furthermore, these findings support 252 Journal of Leukocyte Biology Volume 98, August
5 Petrasek et al. Hepatocyte-derived uric acid and ATP in liver inflammation Figure 4. ATP and soluble uric acid derived from damaged primary murine hepatocytes trigger release of IL-1b and TNF-a from liver immune cells. (A) Primary murine hepatocytes were in vitro exposed to 900 mm ethanol for 2 hours, followed by washing and by postexposure incubation in alcohol- and serum-free media for 12 hours. Afterward, hepatocyte supernatant was collected, LPS (100 ng/ml), uricase (0.1 unit/ml), or apyrase (1 unit/ml) was added, and the hepatocyte supernatant was used to replace 75% of media of nonstimulated LMNCs that had already been seeded in a 96-well plate for 1 hour. Five hours after addition of hepatocyte media to LMNCs, IL-1b and TNF-a were measured in the supernatant. (B) LMNCs were seeded on 96-well plates. After 1 hour, media were replaced with a new media, admixed with cell-free supernatant from HS or control hepatocytes at indicated ratios, and LPS (100 ng/ml), uricase (0.1 unit/ml), or apyrase (1 unit/ml) was added. Five hours afterward, supernatants were collected, and IL-1b was measured. (C) LMNCs were seeded on 96-well plates. Cells were primed with LPS (10 ng/ml). After 30 minutes, IL-1b-neutralizing antibody or anti-goat IgG antibody (0.25 mg/ml each) was added. After 30 minutes, cells were stimulated with ATP (5 mm). Six hours afterward, supernatants were collected and analyzed for IL-1b and TNF-a by ELISA. Numbers in the graphs indicate P values. *P, 0.05 versus baseline. our previous findings showing that low concentrations of IL-1b may have a major impact on inflammatory signaling in ALD [2]. Overall, these data suggested the presence of cross-talk between alcohol-damaged hepatocytes and liver immune cells and demonstrated that inflammatory activation of LMNCs is augmented by hepatocyte-derived uric acid and ATP and is dependent on a feed-forward induction via inflammasome activation and release of IL-1b. Liver inflammation in ALD requires NLRP3, a sensor of metabolic danger Our data indicated that uric acid and ATP, released from damaged hepatocytes, are required for activation of liver immune cells. The Volume 98, August 2015 Journal of Leukocyte Biology 253
6 mechanism by which uric acid and ATP activate inflammation requires the NLRP3 inflammasome [20]. With the use of the NLRP3, immune cells recognize endogenous danger signals, including ATP, uric acid, and crystalline substances, and trigger activation of caspase-1 and maturation of IL-1b [9]. Therefore, we evaluated mice lacking NLRP3 and asked whether NLRP3 provides a mechanistic link among hepatocyte-derived danger signals, inflammasome activation, and liver inflammation in ALD. Deficiency of NLRP3 prevented alcohol-induced activation of the inflammasome in the liver, as indicated by the diminished levels of the cleaved p10 fragment of caspase-1 (Fig. 5A) and cleaved IL-1b in the liver and in the serum (Fig. 5B and C) compared with WT mice. Active, secreted IL-1b up-regulates inflammatory cytokines, including TNF-a, promotes steatosis, and sensitizes hepatocyte to cytotoxicity induced by TNF-a [2, 12, 19, 21]. Accordingly, lack of activation of inflammasome and IL-1b in NLRP3-deficient mice was accompanied by protection from liver inflammation,steatosis,anddamage,as indicated by the absence of alcohol-induced up-regulation of TNF-a and IL-1b in the liver and in the serum (Fig. 5C), improved findings on H&E and amelioration of an ALT increase in the serum (Fig. 5D), absent cleavage of proapoptotic caspase-3 in the liver (Fig. 5E), and decreased lipid accumulation (Fig. 5F). The extent of protection from liver inflammation, injury, and steatosis in NLRP3-knockout mice was comparable with that observed previously in mice deficient in caspase-1, the effector molecule of inflammasomes [2], indicating that NLRP3 is a major activator of the inflammasome in ALD. Taken together, these data further indicated that endogenous danger signals, uric acid, and ATP are involved in inflammatory cross-talk between hepatocytes and immune Figure 5. Deficiency of NLRP3 ameliorates alcohol-induced liver inflammation. WT or NLRP3-knockout (KO) mice were fed control (Pair-fed) or alcohol (EtOH) diet. After 4 weeks, we evaluated cleavage of casp-1 (Casp-1; A) by use of an antibody that identifies full-length proform (short exposure) and cleaved form (long exposure) and cleavage of IL-1b in the liver (B). Levels of total IL-1b and TNF-a (C) in the serum and in the liver were evaluated by use of specific ELISA. Liver damage was assessed by liver histology and serum ALT (D) or by the extent of cleavage of caspase-3 (E). Oil Red O staining was performed, and liver triglycerides were measured to evaluate steatosis (F); n = 7 15 (ethanol-fed/genotype); 4 7 (pair-fed/genotype). Numbers in the graphs indicate P values. *P, 0.05 versus baseline. Original magnification, Journal of Leukocyte Biology Volume 98, August
7 Petrasek et al. Hepatocyte-derived uric acid and ATP in liver inflammation cells in ALD and that their effect is mediated via the NLRP3 inflammasome. In this study, we found that alcohol-induced hepatocyte damage and hepatocyte-derived danger molecules, uric acid and ATP, triggered secretion of IL-1b from liver immune cells. In light of our previous results showing that inflammasomedependent IL-1b is crucially involved in the pathogenesis of ALD [22], our data strongly support the hypothesis that hepatocyte-derived uric acid and ATP represent an endogenous metabolic danger signal for activation of inflammasome/ IL-1b in ALD. Our results suggest that uric acid and ATP represent the 2nd signal in inflammasome activation, thus enabling the 1st signal, represented by gut-derived LPS and dependent on Kupffer cell-specific TLR4 signaling [3 8], to initiate liver inflammation. To the best of our knowledge, thisconcepthasnotbeenreportedpreviouslyinthecontext of ALD. Our results strongly support several novel aspects of inflammation in ALD. First, alcohol-induced metabolic stress results in release of the endogenous danger signals, uric acid and extracellular ATP, from hepatocytes, leading to inflammasome activation in liver immune cells. Second, activation of innate immune cells is triggered by alcohol-induced metabolic danger signals, supporting the hypothesis that outcomes of metabolic signaling in the liver may trigger innate-immune signaling in ALD. Third, development of liver inflammation in ALD seems to be dependent, not only on gutderived LPS but also on endogenous ligands released from damaged hepatocytes and cellular cross-talk between hepatocytes and immune cells. Our findings of protection from liver inflammation in alcohol-fed mice deficient in NLRP3 are consistent with areportbyshulgaandpastorino[23],demonstratingthat activation of murine Kupffer cells by ethanol requires NLRP3, a ligand-sensing component of inflammasomes that detects ATP and uric acid [9]. The requirement for NLRP3 in the development of ALD is supported further by the study of Cui et al. [24], who demonstrated protection from liver inflammation, damage, and steatosis in NLRP3-deficient mice subjected to acute-on-chronic exposure to ethanol. A report by DeSantis et al. [25] demonstrated that mice deficient in NLRP3 were not protected from alcohol-induced liver inflammation. Our previous data demonstrated the requirement for ASC, an adaptor molecule immediately downstream of NLRP3, for activation of inflammasome in ALD. The indispensability of ASC in ALD narrows the choice of inflammasome activators to 2 molecules: NLRP3, supported by these data and reports by Shulga and Pastorino [23] and Cui et al. [24], or an alternative intracellular ligand receptor, AIM2 [9], implied by the negative findings of DeSantis et al. [25]. AIM2 recognizes cytoplasmic dsdna [9], and its role in ALD has yet to be elucidated. In summary, our data indicate that hepatocytes, damaged by ethanol, release endogenous danger molecules, uric acid, and ATP, which are recognized by liver immune cells as inflammatory signals. The final common pathway of this signaling is most likely represented by the activation of inflammasome, which is required for processing and activation of IL-1b, a key inflammatory cytokine involved in the pathogenesis of alcoholic steatohepatitis [2]. These data may provide the basis for future mechanistic studies, as well as for clinical interventions in liver diseases. AUTHORSHIP J.P., A.I.-V., and G.S. designed the research. J.P., A.I.-V., B.S., A.S., and G.S. performed the research. E.A.K.-J. and K.A.F. contributed new reagents/analytic tools. J.P., A.I.-V., K.K., and G.S. analyzed data. J.P., A.I.-V., A.S., E.A.K.-J., K.A.F., and G.S. wrote the paper. ACKNOWLEDGMENTS This work was supported by Grants AA and AA from the U.S. National Institutes of Health National Institute on Alcohol Abuse and Alcoholism (to G.S.). Core resources, supported by the U.S. Diabetes Endocrinology Research Center (Grant DK32520), were also used. The authors thank Anna Cerny for excellent technical assistance. DISCLOSURES There are no conflicts of interest to declare. REFERENCES 1. Gao, B., Seki, E., Brenner, D. A., Friedman, S., Cohen, J. I., Nagy, L., Szabo, G., Zakhari, S. (2011) Innate immunity in alcoholic liver disease. Am. J. Physiol. Gastrointest. Liver Physiol. 300, G516 G Petrasek, J., Bala, S., Csak, T., Lippai, D., Kodys, K., Menashy, V., Barrieau, M., Min, S. Y., Kurt-Jones, E. A., Szabo, G. (2012) IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice. J. Clin. Invest. 122, Adachi, Y., Moore, L. E., Bradford, B. U., Gao, W., Thurman, R. G. (1995) Antibiotics prevent liver injury in rats following long-term exposure to ethanol. Gastroenterology 108, Adachi, Y., Bradford, B. U., Gao, W., Bojes, H. K., Thurman, R. G. (1994) Inactivation of Kupffer cells prevents early alcohol-induced liver injury. Hepatology 20, Bode, C., Bode, J. C. (2005) Activation of the innate immune system and alcoholic liver disease: effects of ethanol per se or enhanced intestinal translocation of bacterial toxins induced by ethanol? Alcohol. Clin. Exp. Res. 29 ((11): Suppl) 166S 171S. 6. Hritz,I.,Mandrekar,P.,Velayudham,A.,Catalano,D.,Dolganiuc, A.,Kodys,K.,Kurt-Jones,E.,Szabo, G. (2008) The critical role of Toll-like receptor (TLR) 4 in alcoholic liver disease is independentofthecommontlradaptermyd88.hepatology 48, Bala, S., Marcos, M., Gattu, A., Catalano, D., Szabo, G. (2014) Acute binge drinking increases serum endotoxin and bacterial DNA levels in healthy individuals. PLoS ONE 9, e Inokuchi, S., Tsukamoto, H., Park, E., Liu, Z. X., Brenner, D. A., Seki, E. (2011) Toll-like receptor 4 mediates alcohol-induced steatohepatitis through bone marrow-derived and endogenous liver cells in mice. Alcohol. Clin. Exp. Res. 35, Schroder, K., Tschopp, J. (2010) The inflammasomes. Cell 140, Petrasek, J., Dolganiuc, A., Csak, T., Nath, B., Hritz, I., Kodys, K., Catalano, D., Kurt-Jones, E., Mandrekar, P., Szabo, G. (2011) Interferon regulatory factor 3 and type I interferons are protective in alcoholic liver injury in mice by way of crosstalk of parenchymal and myeloid cells. Hepatology 53, Dolganiuc, A., Oak, S., Kodys, K., Golenbock, D. T., Finberg, R. W., Kurt-Jones, E., Szabo, G. (2004) Hepatitis C core and nonstructural 3 proteins trigger Toll-like receptor 2-mediated pathways and inflammatory activation. Gastroenterology 127, Volume 98, August 2015 Journal of Leukocyte Biology 255
8 12. Petrasek, J., Dolganiuc, A., Csak, T., Kurt-Jones, E. A., Szabo, G. (2011) Type I interferons protect from Toll-like receptor 9-associated liver injury and regulate IL-1 receptor antagonist in mice. Gastroenterology 140, e Shi, Y., Evans, J. E., Rock, K. L. (2003) Molecular identification of a danger signal that alerts the immune system to dying cells. Nature 425, Feranchak, A. P., Lewis, M. A., Kresge, C., Sathe, M., Bugde, A., Luby- Phelps, K., Antich, P. P., Fitz, J. G. (2010) Initiation of purinergic signaling by exocytosis of ATP-containing vesicles in liver epithelium. J. Biol. Chem. 285, Schroder, K., Zhou, R., Tschopp, J. (2010) The NLRP3 inflammasome: a sensor for metabolic danger? Science 327, Dinarello, C. A. (2009) Immunological and inflammatory functions of the interleukin-1 family. Annu. Rev. Immunol. 27, Granowitz, E. V., Clark, B. D., Vannier, E., Callahan, M. V., Dinarello, C. A. (1992) Effect of interleukin-1 (IL-1) blockade on cytokine synthesis: I. IL-1 receptor antagonist inhibits IL-1-induced cytokine synthesis and blocks the binding of IL-1 to its type II receptor on human monocytes. Blood 79, Granowitz,E.V.,Vannier,E.,Poutsiaka, D. D., Dinarello, C. A. (1992) Effect of interleukin-1 (IL-1) blockade on cytokine synthesis: II. IL-1 receptor antagonist inhibits lipopolysaccharideinduced cytokine synthesis by human monocytes. Blood 79, Cosgrove, B. D., Cheng, C., Pritchard, J. R., Stolz, D. B., Lauffenburger, D. A., Griffith, L. G. (2008) An inducible autocrine cascade regulates rat hepatocyte proliferation and apoptosis responses to tumor necrosis factor-alpha. Hepatology 48, Tschopp, J., Schroder, K. (2010) NLRP3 inflammasome activation: the convergence of multiple signalling pathways on ROS production? Nat. Rev. Immunol. 10, Miura, K., Kodama, Y., Inokuchi, S., Schnabl, B., Aoyama, T., Ohnishi, H., Olefsky, J. M., Brenner, D. A., Seki, E. (2010) Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice. Gastroenterology 139, e Lippai, D., Bala, S., Petrasek, J., Csak, T., Levin, I., Kurt-Jones, E. A., Szabo, G. (2013) Alcohol-induced IL-1b in the brain is mediated by NLRP3/ASC inflammasome activation that amplifies neuroinflammation. J. Leukoc. Biol. 94, Shulga, N., Pastorino, J. G. (2014) Hexokinase II binding to mitochondria is necessary for Kupffer cell activation and is potentiated by ethanol exposure. J. Biol. Chem. 289, Cui, K., Yan, G., Xu, C., Chen, Y., Wang, J., Zhou, R., Bai, L., Lian, Z., Wei, H., Sun, R., Tian, Z. (2015) Invariant NKT cells promote alcohol-induced steatohepatitis through interleukin-1b in mice. J. Hepatol. pii: S , DeSantis, D. A., Ko, C. W., Liu, Y., Liu, X., Hise, A. G., Nunez, G., Croniger, C. M. (2013) Alcohol-induced liver injury is modulated by Nlrp3 and Nlrc4 inflammasomes in mice. Mediators Inflamm. 2013, KEY WORDS: inflammasome NLRP3 sterile inflammatory response DAMPs liver inflammation 256 Journal of Leukocyte Biology Volume 98, August
IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice
IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice Jan Petrasek,, Evelyn A. Kurt-Jones, Gyongyi Szabo J Clin Invest. 2012;122(10):3476-3489. https://doi.org/10.1172/jci60777.
More informationAlcoholic hepatitis is a drug-induced disorder
Alcoholic hepatitis is a drug-induced disorder Gyongyi Szabo, MD, PhD Professor of Medicine University of Massachusetts Medical School Source: 2 Sobernation.com Clinical Progression of ALD Mortality Acute
More informationJournal club. Lama Nazzal
Journal club Lama Nazzal Background Kidney stone disease affects about 12% of men and 5% of women during their lifetimes in the United States Intrarenal nephrocalcinosis is often asymptomatic, but can
More informationInnate Immunity & Inflammation
Innate Immunity & Inflammation The innate immune system is an evolutionally conserved mechanism that provides an early and effective response against invading microbial pathogens. It relies on a limited
More informationSupplementary information
Supplementary information Supplementary Figure S1: Ex[Ca 2+ ]-induced IL-1ß production of monocytes primed with different TLR ligands IL-1ß release of CD14+ monocytes in response to stimulation for 16
More informationType of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures
Type of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures Type of file: MOV Title of file for HTML: Supplementary Movie 1 Description: NLRP3 is moving along
More informationInnate immunity. Abul K. Abbas University of California San Francisco. FOCiS
1 Innate immunity Abul K. Abbas University of California San Francisco FOCiS 2 Lecture outline Components of innate immunity Recognition of microbes and dead cells Toll Like Receptors NOD Like Receptors/Inflammasome
More informationManagement of alcoholic hepatitis: Implications for options beyond the STOPAH study
Management of alcoholic hepatitis: Implications for options beyond the STOPAH study Gyongyi Szabo, MD, PhD Professor University of Massachusetts Medical School Worcester, MA Cape Town, South Africa 2015
More informationThe liver in poisoning: what can we learn from animal models?
The liver in poisoning: what can we learn from animal models? Stephan Krähenbühl Clinical Pharmacology & Toxicology University Hospital 4031 Basel/Switzerland Kraehenbuehl@uhbs.ch Outcome and causes of
More information2. Innate immunity 2013
1 Innate Immune Responses 3 Innate immunity Abul K. Abbas University of California San Francisco The initial responses to: 1. Microbes: essential early mechanisms to prevent, control, or eliminate infection;
More informationINFLAMMASOME IN INTESTINAL INFLAMMATION AND CANCER. Laura Stronati ENEA - Roma
INFLAMMASOME IN INTESTINAL INFLAMMATION AND CANCER Laura Stronati ENEA - Roma Inflammasome: definition, components and activation TLRs NODs RLRs CLRs PRRs LPS, Flagellin, DNA, RNA, etc) PAMPs DAMPs ATP,
More informationGout and Nucleic Acid Metabolism Vol.33 No
Gout and Nucleic Acid Metabolism Vol.33 No.1 2009 1 1 2 3 in vitro 14 IgM 1 IgM IgM 1 PAMPs Pattern recognition receptors PRRs PRRs PRRs PAMPs Toll Toll-like receptor TLR PAMPs Nod Nod-like receptor NLR
More informationExosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific mirna-122 and sensitize monocytes to LPS
University of Massachusetts Medical School escholarship@umms Open Access Articles Open Access Publications by UMMS Authors 5-14-2015 Exosomes derived from alcohol-treated hepatocytes horizontally transfer
More informationFOR OPTIMAL GUT HEALTH KEMIN.COM/GUTHEALTH
FOR OPTIMAL GUT HEALTH KEMIN.COM/GUTHEALTH ALETA A SOURCE OF 1,3-BETA GLUCANS Aleta is highly bioavailable, offering a concentration greater than 5% of 1,3-beta glucans. Aleta provides a consistent response
More informationGut microbiota, metabolic syndrome, obesity and the nutrient sensor pathways
Gut microbiota, metabolic syndrome, obesity and the nutrient sensor pathways Department of Gastroenterology, Endocrinology & Metabolism Medical University Innsbruck Herbert Tilg Nothing to disclose Fig.
More informationProgrammed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsrna-induced retinal degeneration
Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsrna-induced retinal degeneration The Harvard community has made this article openly available. Please
More informationSupplementary fig. 1. Crystals induce necroptosis does not involve caspases, TNF receptor or NLRP3. A. Mouse tubular epithelial cells were pretreated
Supplementary fig. 1. Crystals induce necroptosis does not involve caspases, TNF receptor or NLRP3. A. Mouse tubular epithelial cells were pretreated with zvad-fmk (10µM) and exposed to calcium oxalate
More informationIntrinsic cellular defenses against virus infection
Intrinsic cellular defenses against virus infection Detection of virus infection Host cell response to virus infection Interferons: structure and synthesis Induction of antiviral activity Viral defenses
More informationRole of Innate Immunity in Control of Adaptive Immunity
Role of Innate Immunity in Control of Adaptive Immunity Innate Immunity The burden of pathogen sensing is placed on the innate immune system Danger hypothesis Missing Self Based on the detection of molecular
More informationMcAb and rhil-2 activated bone marrow on the killing and purging of leukemia cells
Effects of McAb and rhil-2 activated bone marrow on the killing and purging of leukemia cells X.C. Wei, D.D. Yang, X.R. Han, Y.A. Zhao, Y.C. Li, L.J. Zhang and J.J. Wang Institute of hematological research,
More informationSupplementary Figure 1
Supplementary Figure 1 AAV-GFP injection in the MEC of the mouse brain C57Bl/6 mice at 4 months of age were injected with AAV-GFP into the MEC and sacrificed at 7 days post injection (dpi). (a) Brains
More informationIntracellular MHC class II molecules promote TLR-triggered innate. immune responses by maintaining Btk activation
Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining Btk activation Xingguang Liu, Zhenzhen Zhan, Dong Li, Li Xu, Feng Ma, Peng Zhang, Hangping Yao and Xuetao
More informationThe Role of Innate Immunity in Alcoholic Liver Disease
ALCOHOL RESEARCH: Current Reviews The Role of Innate Immunity in ic Liver Disease Laura E. Nagy, Ph.D. Laura E. Nagy, Ph.D., is a professor of molecular medicine at Case Western Reserve University School
More informationCytokines (II) Dr. Aws Alshamsan Department of Pharmaceu5cs Office: AA87 Tel:
Cytokines (II) Dr. Aws Alshamsan Department of Pharmaceu5cs Office: AA87 Tel: 4677363 aalshamsan@ksu.edu.sa Learning Objectives By the end of this lecture you will be able to: 1 Understand the physiological
More informationInflammation: How to Cool the Fire Inside your Gut? REINVENTING DIAGNOSTICS
Inflammation: How to Cool the Fire Inside your Gut? REINVENTING DIAGNOSTICS Future of Healthcare REINVENTING DIAGNOSTICS Inflammation Gut Inflammation Basis of a Healthy
More informationSupplementary Figure S1. Effect of Glucose on Energy Balance in WT and KHK A/C KO
Supplementary Figure S1. Effect of Glucose on Energy Balance in WT and KHK A/C KO Mice. WT mice and KHK-A/C KO mice were provided drinking water containing 10% glucose or tap water with normal chow ad
More informationSupplementary Figure 1: Hsp60 / IEC mice are embryonically lethal (A) Light microscopic pictures show mouse embryos at developmental stage E12.
Supplementary Figure 1: Hsp60 / IEC mice are embryonically lethal (A) Light microscopic pictures show mouse embryos at developmental stage E12.5 and E13.5 prepared from uteri of dams and subsequently genotyped.
More informationIslet viability assay and Glucose Stimulated Insulin Secretion assay RT-PCR and Western Blot
Islet viability assay and Glucose Stimulated Insulin Secretion assay Islet cell viability was determined by colorimetric (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay using CellTiter
More informationSupporting Information
Supporting Information Pang et al. 10.1073/pnas.1322009111 SI Materials and Methods ELISAs. These assays were performed as previously described (1). ELISA plates (MaxiSorp Nunc; Thermo Fisher Scientific)
More informationImmunology Part II. Innate Immunity. 18. April 2018, Ruhr-Universität Bochum Marcus Peters,
Immunology Part II Innate Immunity 18. April 2018, Ruhr-Universität Bochum Marcus Peters, marcus.peters@rub.de Conserved structures of pathogens PAMPs are detected by Pattern Recognition Receptors PRRs
More informationCaspase-11 Activation in Response to Bacterial Secretion Systems That Access the Host Cytosol
University of Pennsylvania ScholarlyCommons Department of Microbiology Perelman School of Medicine 6-6-2013 Caspase-11 Activation in Response to Bacterial Secretion Systems That Access the Host Cytosol
More informationINTRODUCTION. Pranoti Mandrekar, 1 Donna Catalano, Valentina Jeliazkova, and Karen Kodys
Alcohol exposure regulates heat shock transcription factor binding and heat shock proteins 70 and 90 in monocytes and macrophages: implication for TNF- regulation Pranoti Mandrekar, 1 Donna Catalano, Valentina
More informationAccepted Manuscript. Innate immune cells regulate oncoimmunity and cancer development. Ai-Ping Bai, Yuan Guo
Accepted Manuscript Innate immune cells regulate oncoimmunity and cancer development Ai-Ping Bai, Yuan Guo PII: S0016-5085(18)34974-6 DOI: 10.1053/j.gastro.2018.08.057 Reference: YGAST 62119 To appear
More informationCell isolation. Spleen and lymph nodes (axillary, inguinal) were removed from mice
Supplementary Methods: Cell isolation. Spleen and lymph nodes (axillary, inguinal) were removed from mice and gently meshed in DMEM containing 10% FBS to prepare for single cell suspensions. CD4 + CD25
More informationPrimer sequences Target Sequence F Sequence R TNF-α (Tnfa) TCAGCCGATTTGCTATCTCAT A
Supplementary Table 1. Q- and RT-PR primers used in this study. Primer sequences Target Sequence F Sequence R TNF-α (Tnfa) TGGTTTGTTTT GTTTGGGGTTG T hemokine (- motif) ligand 5 (cl5) GTGTTTGTTT TGGTGGTG
More informationBile acids initiate cholestatic liver injury by triggering a hepatic specific inflammatory response. Supplementary Results
Bile acids initiate cholestatic liver injury by triggering a hepatic specific inflammatory response Shi-Ying Cai 1, Xinshou Ouyang 1, Yonglin Chen 1, Carol J. Soroka 1, Juxian Wang 2, Albert Mennone 1,
More informationAlcoholic liver disease (ALD) ranges from simple
HEPATOLOGY, VOL. 64, NO. 6, 2016 AMERICAN ASSOCIATION FOR THE STUDY OFLIVERD I S E ASES IRAKM-Mincle Axis Links Cell Death to Inflammation: Pathophysiological Implications for Chronic Alcoholic Liver Disease
More informationUnderstanding the Role of the Inflammasome in Inflammation
Understanding the Role of the Inflammasome in Inflammation Martha O Brien, Ph.D. Assay Design Group 215 Innate Immune Response in Inflammation LPS flagellin TLR-1/2/6 TLR-4 TLR-5 Adaptor proteins PAMPs,
More informationOsteopontin a bioactive milk protein with immunological properties
Osteopontin a bioactive milk protein with immunological properties Esben S. Sørensen Section for Molecular Nutrition Department of Molecular Biology and Genetics Aarhus University, Denmark Proteins in
More informationMedical Virology Immunology. Dr. Sameer Naji, MB, BCh, PhD (UK) Head of Basic Medical Sciences Dept. Faculty of Medicine The Hashemite University
Medical Virology Immunology Dr. Sameer Naji, MB, BCh, PhD (UK) Head of Basic Medical Sciences Dept. Faculty of Medicine The Hashemite University Human blood cells Phases of immune responses Microbe Naïve
More informationEndocannabinoid-activated Nlrp3 inflammasome in infiltrating macrophages mediates β- cell loss in type 2 diabetes
Endocannabinoid-activated Nlrp3 inflammasome in infiltrating macrophages mediates β- cell loss in type 2 diabetes T Jourdan, G Godlewski, R Cinar, A Bertola, G Szanda, J Liu, J Tam, T Han, B Mukhopadhyay,
More informationAnti-Apoptotic Effects of Cellular Therapy
Anti-Apoptotic Effects of Cellular Therapy Jason Lapetoda 1, Lee K Landeen, PhD 1, George K Michalopoulos, MD 2, Patricia W Bedard, PhD 1 1 Vital Therapies, Inc., San Diego, CA, USA 2 University of Pittsburgh
More informationCD40L TCR IL-12 TLR-L
CD40L B cells plasmacells Neutrophils TCR inkt cells IL-12 Ab production Can inkt cells modulate the cytokine profile of neutrophils? TLR-L CD4+ and CD8+ T cell responses 1. The invariant TCR expressed
More informationAttribution: University of Michigan Medical School, Department of Microbiology and Immunology
Attribution: University of Michigan Medical School, Department of Microbiology and Immunology License: Unless otherwise noted, this material is made available under the terms of the Creative Commons Attribution
More informationSupporting Information
Supporting Information Desnues et al. 10.1073/pnas.1314121111 SI Materials and Methods Mice. Toll-like receptor (TLR)8 / and TLR9 / mice were generated as described previously (1, 2). TLR9 / mice were
More informationToll-like Receptor Signaling
Toll-like Receptor Signaling 1 Professor of Medicine University of Massachusetts Medical School, Worcester, MA, USA Why do we need innate immunity? Pathogens multiply very fast We literally swim in viruses
More informationElaborating ELAD Mechanism of Action and Linking Cell-Based Models to the Clinic
Elaborating ELAD Mechanism of Action and Linking Cell-Based Models to the Clinic September 9, 2017 Patricia W Bedard, Jason Lapetoda, Jagadeesha Dammanahalli, Jessica Van Allen, Susan Lin, Lee Landeen
More informationT-cell activation T cells migrate to secondary lymphoid tissues where they interact with antigen, antigen-presenting cells, and other lymphocytes:
Interactions between innate immunity & adaptive immunity What happens to T cells after they leave the thymus? Naïve T cells exit the thymus and enter the bloodstream. If they remain in the bloodstream,
More informationT-cell activation T cells migrate to secondary lymphoid tissues where they interact with antigen, antigen-presenting cells, and other lymphocytes:
Interactions between innate immunity & adaptive immunity What happens to T cells after they leave the thymus? Naïve T cells exit the thymus and enter the bloodstream. If they remain in the bloodstream,
More informationImpact factor: Reporter:4A1H0019 Chen Zi Hao 4A1H0023 Huang Wan ting 4A1H0039 Sue Yi Zhu 4A1H0070 Lin Guan cheng 4A1H0077 Chen Bo xuan
Curcumin Protects Neonatal Rat Cardiomyocytes against High Glucose-Induced Apoptosis via PI3K/Akt Signalling Pathway Wei Yu,1,2 Wenliang Zha,1 Zhiqiang Ke,1 Qing Min,2 Cairong Li,1 Huirong Sun,3 and Chao
More informationProcaspase-3. Cleaved caspase-3. actin. Cytochrome C (10 M) Z-VAD-fmk. Procaspase-3. Cleaved caspase-3. actin. Z-VAD-fmk
A HeLa actin - + + - - + Cytochrome C (1 M) Z-VAD-fmk PMN - + + - - + actin Cytochrome C (1 M) Z-VAD-fmk Figure S1. (A) Pan-caspase inhibitor z-vad-fmk inhibits cytochrome c- mediated procaspase-3 cleavage.
More informationLPS LPS P6 - + Supplementary Fig. 1.
P6 LPS - - - + + + - LPS + + - - P6 + Supplementary Fig. 1. Pharmacological inhibition of the JAK/STAT blocks LPS-induced HMGB1 nuclear translocation. RAW 267.4 cells were stimulated with LPS in the absence
More informationSupplementary Table 1. Antibodies and dilutions used in the immunohistochemical study
Supplementary Table 1. Antibodies and dilutions used in the immunohistochemical study Antigen Species Clone Source Dilution Insulin Guinea pig - Dako, Carpinteria, 1:200 Glucagon Rabbit - Dako, Carpinteria,
More informationTLR2/MyD88/NF-kB Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection
TLR2/MyD88/NF-kB Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection Jesus Segovia 1., Ahmed Sabbah 1., Victoria Mgbemena 1,
More informationChapter 13: Cytokines
Chapter 13: Cytokines Definition: secreted, low-molecular-weight proteins that regulate the nature, intensity and duration of the immune response by exerting a variety of effects on lymphocytes and/or
More informationIdentification of Microbes
Identification of Microbes Recognition by PRR (pattern recognition receptors) Recognize conserved molecular patterns on microbes called pathogen associated molecular patterns (PAMPs) which are not present
More informationNonalcoholic fatty liver disease (NAFLD) is
Fatty Acid and Endotoxin Activate Inflammasomes in Mouse Hepatocytes that Release Danger Signals to Stimulate Immune Cells Timea Csak, Michal Ganz, Justin Pespisa, Karen Kodys, Angela Dolganiuc, and Gyongyi
More informationExosomes function in antigen presentation during an in vivo Mycobacterium tuberculosis infection
Exosomes function in antigen presentation during an in vivo Mycobacterium tuberculosis infection Victoria L. Smith, Yong Cheng, Barry R. Bryant and Jeffrey S. Schorey Supplementary Figure 1: Unprocessed
More informationA Yersinia-Secreted Effector Protein Promotes Virulence by Preventing Inflammasome Recognition of the Type III Secretion System
Cell Host & Microbe, Volume 7 Supplemental Information A Yersinia-Secreted Effector Protein Promotes Virulence by Preventing Inflammasome Recognition of the Type III Secretion System Igor E. Brodsky, Noah
More informationSystemic inflammation after myocardial infarction
Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2013 Systemic inflammation after myocardial infarction Rudiger, Alain DOI:
More informationExcessive alcohol consumption leads to alcoholic
Hepatology Communications, VOL. 2, NO. 11, 2018 FXR and TGR5 Agonists Ameliorate Liver Injury, Steatosis, and Inflammation After Binge or Prolonged Alcohol Feeding in Mice Arvin Iracheta-Vellve, 1 Charles
More information12/10/2009. Department of Pathology, Case Western Reserve University. Mucosal Cytokine Network in IBD
Cytokine-Mediated Inflammation in IBD Theresa T. Pizarro Department of Pathology, Case Western Reserve University Mucosal Cytokine Network in IBD Andoh, et al., 2008 1 Interleukin-1 (IL-1) Family Cytokine
More informationSupporting Information
Supporting Information Chen et al. 10.1073/pnas.0807991106 SI Methods Sucrose Gradient Fractionation. Fractionation by sucrose gradient was performed as described by Gasparini et al. [(2001) J Neurosci
More informationActivation of inflammatory response by fungal cell wall components and toxins
Finnish Institute of Occupational Health Activation of inflammatory response by fungal cell wall components and toxins Sampsa Matikainen Innate Immunity Research Group Unit of Immunotoxicology Finnish
More informationRole of Innate Immunity in Hepatitis B Virus Infection Adam J. Gehring, Ph.D.
Role of Innate Immunity in Hepatitis B Virus Infection Adam J. Gehring, Ph.D. Biology Lead Toronto Centre for Liver Disease Toronto General Hospital Research Institute University Health Network (UHN) HBV
More informationSUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION doi:10.1038/nature11429 S1a 6 7 8 9 Nlrc4 allele S1b Nlrc4 +/+ Nlrc4 +/F Nlrc4 F/F 9 Targeting construct 422 bp 273 bp FRT-neo-gb-PGK-FRT 3x.STOP S1c Nlrc4 +/+ Nlrc4 F/F casp1
More informationOverview of the immune system
Overview of the immune system Immune system Innate (nonspecific) 1 st line of defense Adaptive (specific) 2 nd line of defense Cellular components Humoral components Cellular components Humoral components
More informationACTIVATION AND EFFECTOR FUNCTIONS OF CELL-MEDIATED IMMUNITY AND NK CELLS. Choompone Sakonwasun, MD (Hons), FRCPT
ACTIVATION AND EFFECTOR FUNCTIONS OF CELL-MEDIATED IMMUNITY AND NK CELLS Choompone Sakonwasun, MD (Hons), FRCPT Types of Adaptive Immunity Types of T Cell-mediated Immune Reactions CTLs = cytotoxic T lymphocytes
More informationCell Quality Control. Peter Takizawa Department of Cell Biology
Cell Quality Control Peter Takizawa Department of Cell Biology Cellular quality control reduces production of defective proteins. Cells have many quality control systems to ensure that cell does not build
More informationFigure S1. Western blot analysis of clathrin RNA interference in human DCs Human immature DCs were transfected with 100 nm Clathrin SMARTpool or
Figure S1. Western blot analysis of clathrin RNA interference in human DCs Human immature DCs were transfected with 100 nm Clathrin SMARTpool or control nontargeting sirnas. At 90 hr after transfection,
More informationCommensal Bacteria, Toll-like Receptors and Intestinal Injury. Journal Club December 16, 2004
Commensal Bacteria, Toll-like Receptors and Intestinal Injury Journal Club December 16, 2004 Gut-Commensal Interactions Nutrient metabolism Tissue development Resistance to colonization with pathogens
More informationCytokines modulate the functional activities of individual cells and tissues both under normal and pathologic conditions Interleukins,
Cytokines http://highered.mcgraw-hill.com/sites/0072507470/student_view0/chapter22/animation the_immune_response.html Cytokines modulate the functional activities of individual cells and tissues both under
More informationCrucial role for human Toll-like receptor 4 in the development of contact allergy to nickel
Supplementary Figures 1-8 Crucial role for human Toll-like receptor 4 in the development of contact allergy to nickel Marc Schmidt 1,2, Badrinarayanan Raghavan 1,2, Verena Müller 1,2, Thomas Vogl 3, György
More informationIntegrin CD11b negatively regulates TLR-triggered inflammatory responses by. activating Syk and promoting MyD88 and TRIF degradation via cbl-b
Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting MyD88 and TRIF degradation via cbl-b Chaofeng Han, Jing Jin, Sheng Xu, Haibo Liu, Nan Li, and Xuetao
More informationSUPPLEMENTARY INFORMATION
Supplemental Figure 1. Furin is efficiently deleted in CD4 + and CD8 + T cells. a, Western blot for furin and actin proteins in CD4cre-fur f/f and fur f/f Th1 cells. Wild-type and furin-deficient CD4 +
More informationThe death receptors: signaling and modulation
The death receptors: signaling and modulation 1 1 The extrinsic cell death pathway 2 Nat Rev Drug Discov. 2008 Dec;7(12):1001-12. 2 Death receptors Belong to the tumor necrosis factor (TNF) receptor gene
More informationSupplementary Figure 1. DNA methylation of the adiponectin promoter R1, Pparg2, and Tnfa promoter in adipocytes is not affected by obesity.
Supplementary Figure 1. DNA methylation of the adiponectin promoter R1, Pparg2, and Tnfa promoter in adipocytes is not affected by obesity. (a) Relative amounts of adiponectin, Ppar 2, C/ebp, and Tnf mrna
More informationSupplementary data Supplementary Figure 1 Supplementary Figure 2
Supplementary data Supplementary Figure 1 SPHK1 sirna increases RANKL-induced osteoclastogenesis in RAW264.7 cell culture. (A) RAW264.7 cells were transfected with oligocassettes containing SPHK1 sirna
More informationBasis of Immunology and
Basis of Immunology and Immunophysiopathology of Infectious Diseases Jointly organized by Institut Pasteur in Ho Chi Minh City and Institut Pasteur with kind support from ANRS & Université Pierre et Marie
More informationIntroduction to pathology lecture 5/ Cell injury apoptosis. Dr H Awad 2017/18
Introduction to pathology lecture 5/ Cell injury apoptosis Dr H Awad 2017/18 Apoptosis = programmed cell death = cell suicide= individual cell death Apoptosis cell death induced by a tightly regulated
More informationSupplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice.
Supplementary Figures: Supplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice. Male apoe -/- mice were fed a high-fat diet for 8 weeks, and given PBS (model group) or
More informationcolorimetric sandwich ELISA kit datasheet
colorimetric sandwich ELISA kit datasheet For the quantitative detection of human TNF-alpha in serum, plasma and cell culture supernatants. general information Catalogue Number Product Name Species cross-reactivity
More informationA Hepatocyte Growth Factor Receptor (Met) Insulin Receptor hybrid governs hepatic glucose metabolism SUPPLEMENTARY FIGURES, LEGENDS AND METHODS
A Hepatocyte Growth Factor Receptor (Met) Insulin Receptor hybrid governs hepatic glucose metabolism Arlee Fafalios, Jihong Ma, Xinping Tan, John Stoops, Jianhua Luo, Marie C. DeFrances and Reza Zarnegar
More informationNASH Bench to Bedside
NASH Bench to Bedside October 2006 Anna Mae Diehl, M.D. Gastroenterology Division Duke University NonAlcoholic Fatty Liver Disease Common ~1/4-1/3 1/3 US adults Outcome highly variable Course indolent
More informationChapter 32. Non specific (Innate) Host Resistance ( 비특이적 ( 내재 ) 숙주방어 )
Chapter 32 Non specific (Innate) Host Resistance ( 비특이적 ( 내재 ) 숙주방어 ) Host Resistance Overview Immune system ( 면역계 ) Composed of widely distributed cells, tissues, and organs Recognizes foreign substances
More informationStructure and Function of Antigen Recognition Molecules
MICR2209 Structure and Function of Antigen Recognition Molecules Dr Allison Imrie allison.imrie@uwa.edu.au 1 Synopsis: In this lecture we will examine the major receptors used by cells of the innate and
More informationMasmudur M. Rahman and Grant McFadden* Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610
JOURNAL OF VIROLOGY, Dec. 2011, p. 12505 12517 Vol. 85, No. 23 0022-538X/11/$12.00 doi:10.1128/jvi.00410-11 Copyright 2011, American Society for Microbiology. All Rights Reserved. Myxoma Virus Lacking
More informationSupplemental Figure 1 ELISA scheme to measure plasma total, mature and furin-cleaved
1 Supplemental Figure Legends Supplemental Figure 1 ELISA scheme to measure plasma total, mature and furin-cleaved PCSK9 concentrations. 4 Plasma mature and furin-cleaved PCSK9s were measured by a sandwich
More informationCHAPTER I INTRODUCTION. Nowadays chronic kidney disease (CKD) becomes one. of the most common diseases found in the population.
CHAPTER I INTRODUCTION I.1 Background Nowadays chronic kidney disease (CKD) becomes one of the most common diseases found in the population. Based on community survey that is held by PERNEFRI (Perhimpunan
More informationSUPPLEMENTAL INFORMATION
SUPPLEMENTAL INFORMATION EXPERIMENTAL PROCEDURES Tryptic digestion protection experiments - PCSK9 with Ab-3D5 (1:1 molar ratio) in 50 mm Tris, ph 8.0, 150 mm NaCl was incubated overnight at 4 o C. The
More informationInnate immunity as a therapeutic target in IBD. Elke Cario Division of Gastroenterology & Hepatology University Hospital of Essen Essen, Germany
Innate immunity as a therapeutic target in IBD Elke Cario Division of Gastroenterology & Hepatology University Hospital of Essen Essen, Germany The intestinal mucosa must rapidly recognize luminal pathogens
More informationCircadian pathways as a mediator between alcohol and liver disease
Circadian pathways as a mediator between alcohol and liver disease Shannon M. Bailey Department of Pathology University of Alabama at Birmingham Circadian Rhythm in GI Health & Disease Rush University
More informationProtection against doxorubicin-induced myocardial dysfunction in mice by cardiac-specific expression of carboxyl terminus of hsp70-interacting protein
Protection against doxorubicin-induced myocardial dysfunction in mice by cardiac-specific expression of carboxyl terminus of hsp70-interacting protein Lei Wang 1, Tian-Peng Zhang 1, Yuan Zhang 2, Hai-Lian
More informationSupporting Information
Supporting Information van der Windt et al. 10.1073/pnas.1221740110 SI Materials and Methods Mice and Reagents. C57BL/6 and major histocompatibility complex class I-restricted OVA-specific T-cell receptor
More informationMANUAL IL-1alpha (mouse) ELISA Kit Cat. No. AG-45B-0003-KI01 [Interleukin-1 alpha (mouse) ELISA Kit]
MANUAL IL-1alpha (mouse) ELISA Kit [Interleukin-1 alpha (mouse) ELISA Kit] For research use only. Not for diagnostic use Version 1 (March-5-2013) Cat. No. AG-45B-0003-KI01 www.adipogen.com Table of Contents
More informationInnate Immunity. Chapter 3. Connection Between Innate and Adaptive Immunity. Know Differences and Provide Examples. Antimicrobial peptide psoriasin
Chapter Know Differences and Provide Examples Innate Immunity kin and Epithelial Barriers Antimicrobial peptide psoriasin -Activity against Gram (-) E. coli Connection Between Innate and Adaptive Immunity
More informationIL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis. in mice
IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice Jan Petrasek, Shashi Bala, Timea Csak, Dora Lippai, Karen Kodys, Victoria Menashy, Matthew Barrieau, So-Yun
More informationRichard S. Kornbluth, M.D., Ph.D.
Treatment of established tumors with peritumoral injections of CD40 ligand (CD40L), CpG, poly(i:c), and extracellular ATP in murine models Richard S. Kornbluth, M.D., Ph.D. Disclosure: Richard Kornbluth
More informationSupplementary Figure 1 Protease allergens induce IgE and IgG1 production. (a-c)
1 Supplementary Figure 1 Protease allergens induce IgE and IgG1 production. (a-c) Serum IgG1 (a), IgM (b) and IgG2 (c) concentrations in response to papain immediately before primary immunization (day
More information