Sa1736 Macrophage Subtypes Disrupt the Epithelium via Deregulated Tight Junctions and Induction of...
1
AGA Abstracts margin in the same patients with Montreal B2 phenotype Crohn's disease. Isolated muscle cells were used to prepare protein lysates, RNA or placed into primary cell cultures. Ex vivo sections and cultured cells were treated with 20 ng/ml IL-6 in the presence and absence of the Jak inhibitor, tofacitinib (10 μM). Phosphorylated isoforms of Jak1(Y1022/1023), Jak2 (Y1007/1008), STAT3(S727) and STAT3(Y705) were measured by immunoblot analysis and by immunofluorescence. Results: In ex vivo sections of intestine muscle cells or isolated cells, levels of phosphorylated Jak1 and Jak2 were increased compared to those in the normal margin in the same patient. Treatment of cultured muscle cells isolated from normal margin with IL-6 elicited the same pattern of increased STAT3(S727) and decreased STAT3(Y705) phosphorylation that was seen in muscle cells in strictured intestine. Tofacitinib selectively inhibited IL-6-induced phosphorylation of Jak1 but not Jak2. In IL-6 treated cells, the high levels of STAT3(S727), low levels STAT3(Y705) phosphorylation, and high TGF-β1 and collagen IαI levels of expression that are characteristic of strictured intestine were restored to that of normal intestine with low levels of STAT3(S727), high levels of STAT3(Y705) phosphorylation and low TGF-β1 and collagen IαI expression. Conclusions: Tofacitinib selectively inhibits Jak1 activity in human intestinal muscle. The effects of tofacitinib on IL- 6-dependent STAT3 activity and increased expression of profibrotic TGF-β1 and collagen IαI suggests tofacitinib may be useful in susceptible patients expressing a Montreal B2 fibrostenotic phenotype of disease. Supported by NIH-DK49691 Sa1734 Pregnane X Receptor Agonists Enhance Intestinal Epithelial Wound Healing and Repair of the Intestinal Barrier Following the Induction of Experimental Colitis Joshua D. Terc, Ashleigh Hansen, Laurie Alston, Simon A. Hirota The intestinal epithelial barrier plays a key role in the maintenance of homeostasis within the gastrointestinal tract. Barrier dysfunction, leading to increased epithelial permeability, is associated with the inflammatory bowel diseases (IBD). It is thought that the increased permeability in patients with IBD may be driven by alterations in the epithelial wound healing response. To this end considerable study has been undertaken to identify signaling pathways that may accelerate intestinal epithelial wound healing and normalize the barrier dysfunction observed in IBD. In the current study we examined the role of the pregnane X receptor (PXR) in modulating the intestinal epithelial wound healing response. Mutations and reduced mucosal expression of the PXR are associated with IBD, and others have reported that PXR agonists can dampen intestinal inflammation. Furthermore, stimulation of the PXR has been associated with increased cell migration and proliferation, two of the key processes involved in wound healing Thus, we sought to test the hypothesis that PXR agonists enhance intestinal epithelial repair. To study wound healing in vitro, Caco-2 intestinal epithelial cells were seeded in Ibidi wound assay inserts and grown to confluence. Wound closure was assessed at 12 and 24 hours following treatment with PXR agonists rifaximin, rifampicin and SR12813. To study intestinal epithelial healing in vivo, mice were treated dextran sulphate sodium (DSS; 2.5% w/v in drinking water) for 7 days and allowed to recover for an additional 7 days on normal drinking water. During the recovery phase, mice were treated with a rodent specific PXR agonist, pregnenolone 16α-carbonitrile (PCN), or vehicle by oral garage. FITC-dextran flux was used to assess in vivo permeability after the recovery. In agreement with our hypothesis, stimulation of Caco-2 cells with rifaximin, rifampicin and SR12813, all potent agonists of the PXR, significantly increased wound closure. This effect was driven by p38 MAP kinase-dependent cell migration, and occurred in the absence of cell proliferation. Treating mice with PCN, a potent rodent specific PXR agonist, attenuated the intestinal barrier dysfunction observed in the DSS model of experimental colitis, an effect that occurred independent of its known anti-inflammatory effects. Taken together our data indicate that the activation of the PXR can enhance intestinal epithelial repair and suggest that targeting the PXR may help to normalize intestinal barrier dysfunction observed in patients with IBD. Furthermore, our data provide additional insight into the potential mechanisms through which rifaximin elicits its clinical efficacy in the treatment of IBD. Sa1735 IL-17A Modulates the TNF-α-Induced Expression of Inflammatory Bowel Disease Susceptibility Genes in Intestinal Epithelial Cells Matthias Friedrich, Julia Diegelmann, Stephan Brand Background: In contrast to anti-TNF-α antibodies, anti-IL-17A antibodies lacked clinical efficacy in a trial with patients suffering from Crohn's disease (CD). Therefore, we analyzed how IL-17A modulates the inflammatory response elicited by TNF-α in intestinal epithelial cells (IEC). Methods: Target mRNA levels in IEC and colonic biopsies were assessed by RNA microarray and qRT-PCR. Signaling pathways were analyzed using receptor neutralization and pharmacological inhibitors. Target protein levels were determined by immunoblotting. Results: Microarray analysis demonstrated that IL-17A alone is a weak inducer of gene expression in IEC (29 regulated transcripts), but significantly affected the TNF-α-induced expression of 547 genes, with a strong amplification of proinflammatory chemokines and cytokines (>200-fold increase of CCL20, CXCL1 and CXCL8). Interestingly, IL-17A differen- tially modulated the TNF-α-induced expression of several IBD susceptibility genes in IEC (increase of JAK2 mRNA, decrease of FUT2, ICOSLG, ICAM1, LTB, and SMAD3 mRNA). For ICOSLG and ICAM1 mRNA, we demonstrated that this is dependent on NF-κB signaling. Negative regulation of ICAM-1 by IL-17A was verified on protein level. The biological significance of these findings is emphasized by inflamed lesions of IBD patients demonstrating increased ICAM1 (P=0.028) and JAK2 (P=0.002) transcript levels. Conclusions: We demon- strates the modulation of IBD susceptibility gene mRNA as a novel important property of IL-17A. Given our microarray data demonstrating a low effect of sole IL-17A stimulation on IEC gene expression, this provides an important explanation for the lack of clinical efficacy of IL-17A neutralization. S-284 AGA Abstracts Sa1736 Macrophage Subtypes Disrupt the Epithelium via Deregulated Tight Junctions and Induction of Apoptosis Donata Lissner, Michael Schumann, Arvind Batra, Lea I. Kredel, Anja A. Kuehl, Britta Siegmund Background We have shown that pro-inflammatory macrophage subtypes, as found in the lamina propria of patients with Crohns disease (CD), are capable of disrupting the epithelial barrier. The exact mechanism has not been described yet. Methods Unpolarized macrophages (M0) were isolated from human peripheral blood and subsequently polarized into M1- and M2-macrophages. The effect of these cells types on epithelial resistance and thus integrity was analysed after co-culture with different epithelial cell lines (Caco-2, T-84, HT-29/B6). Tight junction proteins (claudin-1 and -2, JAM-1, ZO-1, E-cadherin) and caspase-3 and -8 were analysed by immunostaining and western blot. Results Compared to regulatory M2- macrophages, pro-inflammatory M0- and M1 macrophages lead to a massive decrease in epithelial resistance. Mainly, this is due to deregulation of tight junction proteins: The pore- forming protein claudin-2 is significantly up-regulated in M0- and M1-exposed epithelial cells, whereas E-cadherin tends to be decreased in these conditions and increased in M2- exposed cells. Additionally, increased caspase-3 in M0-treated epithelial cells reveals induc- tion of apoptosis as a further mechanism. Here, elevated amounts of active caspase-8 suggest death receptor mediated apoptosis, rather than via mitochondrial regulation. Conclusion The disrupting effect of pro-inflammatory macrophages, as found in the lamina propria of patients with CD, on the epithelial barrier is due to deregulated tight junction proteins and induction of death receptor mediated apoptosis. Sa1737 Osteoprotegerin: A Novel Alarmin in Colonic Epithelial Cells? Raghunath Ramanarasimhaiah, Marina L. Fernandez, Anthony Vella, Francisco A. Sylvester Background: Osteoprotegerin (OPG) blocks osteoclast formation, is involved in metastatic cancer and may have immune regulatory functions. We have reported that fecal OPG is significantly increased in active colonic inflammatory bowel disease (IBD). We hypothesized that colonic epithelial cells synthesize and secrete OPG into the colonic lumen. Aim: To characterize OPG expression in colonic epithelial cells. Methods: We examined the expres- sion of OPG in colonic endoscopic biopsies from children with normal histology and with IBD and in subcellular compartments of the colonic epithelial cell line Caco-2. We used immunohistochemistry (IHC), immunofluorescence (IF) paired with confocal microscopy, Western blot (WB) and immunoprecipitation followed by mass spectrometry (Mass Spec) (primary antibody Cat# IMG103A, Imgenex or Cat# ALX-210-945, Enzo), and ELISA (R& D Systems), as appropriate. OPG mRNA expression was examined with quantitative PCR (qPCR). Recombinant human cytokines were obtained from MBL International Corporation or Cell Signaling Technology. Results: We studied 21 biopsies of children with IBD and 11 with normal histology (CTRL). We observed expression of OPG in lamina propria mononuclear cells (LPMC), but the strongest OPG expression was in colonic epithelial cells. Signal was observed in LPMC only in the cytoplasm, but colonic epithelial cells had signal both in the nucleus and cytoplasm in CTRL. In IBD, nuclear OPG was no longer present in epithelial cells, while strong signal persisted in the cytoplasm. We then studied Caco-2 cells as a model for colonic epithelial cells. At baseline, Caco-2 cells secreted OPG into the medium (mean ± SD, 6.2 ± 0.5 ng/mL OPG. Interleukin (IL)-1β (0.3 ng/mL for 24hrs) increased OPG secretion 2-fold (12.4 ± 0.8 ng/mL; p <0.001); OPG mRNA also increased 6-fold compared to control (p<0.001). TNF-α (100 ng/mL) similarly increased OPG expres- sion, but IL-4 and IL-18 had no effect. IF showed a "starry sky" pattern of OPG in the cytoplasm and densely in the nucleus. Presence of OPG in the nucleus was confirmed by WB, ELISA and Mass Spec of nuclear extracts. Conclusion: We confirm for the first time the presence of nuclear OPG in colonic epithelial cells that is mobilized during inflammation, a pattern akin to IL-33. Our observations suggest that OPG may be an alarmin activated during inflammation in IBD. Sa1738 Paracrine Activity of Necrotic Epithelial IL-1α Mediates Non-Immune Cell Interactions and Amplification of Intestinal Inflammation Melania Scarpa, Tammy M. Sadler, Gail West, Claudio Fiocchi, Eleni Stylianou Introduction Excessive intestinal epithelial cell death is a recognized feature of Inflammatory Bowel Disease (IBD). However, information on damage-associated molecular patterns (DAMPs), the products of necrosis, and on their function in IBD, is limited. We have shown previously that interleukin-1 alpha (IL-1α), is a DAMP produced by necrotic intestinal epithelial cells that activates human intestinal fibroblasts (HIF) cytokine production and exacerbates intestinal injury. We aimed to determine whether intestinal epithelial-fibroblast interactions depend on IL-1α that is i) produced as a DAMP by intestinal epithelial cells, ii) secreted by live epithelial cells in response to proinflammatory or NLRP3 inflammasome stimuli, iii) endogenous to HIF. Materials and Methods Small interfering RNA (siRNA) targeting human IL-1α (ON-TARGET plus SMARTpool, Dharmacon) or a scrambled control siRNA were transfected into HT29 or HIF by nucleofection (Amaxa) for 48 hrs. Transfected or control HIF were incubated with HT29 necrotic cell lysates (NCL, 1/20 dilution in growth medium) prepared from cells transfected with IL-1α siRNA or control HT29 cells for 24hrs. Supernatants were collected and analyzed for IL-6 and IL-8 by ELISA. The levels of IL-1α and β-actin mRNA were determined by RT-PCR. HT29 or human monocytes were incubated with ultrapure LPS (10ng/ml-1μg/ml) followed by incubation for 24hrs with the proinflamma- tory cytokine TNFα (100ng/ml), or with soluble or particulate activators of the NLRP3 inflammasome ATP (5mM), or monsodium urate crystals (MSU, 0.1-0.6 ug/ml). Whole cell lysates and supernatants were prepared and levels of IL-1α, IL-1β and IL-8 quantified by ELISA. Results NC lysates prepared from HT29 cells in which IL-1α was depleted resulted in impaired production of IL-6 and IL-8 by HIF. These cytokine levels were unaffected by transfection of IL-1α siRNA in HIF and were unchanged by transfection of the siRNA control.
Transcript of Sa1736 Macrophage Subtypes Disrupt the Epithelium via Deregulated Tight Junctions and Induction of...
AG
AA
bst
ract
smargin in the same patients with Montreal B2 phenotype Crohn's disease. Isolated musclecells were used to prepare protein lysates, RNA or placed into primary cell cultures. Ex vivosections and cultured cells were treated with 20 ng/ml IL-6 in the presence and absence ofthe Jak inhibitor, tofacitinib (10 μM). Phosphorylated isoforms of Jak1(Y1022/1023), Jak2(Y1007/1008), STAT3(S727) and STAT3(Y705) were measured by immunoblot analysis andby immunofluorescence. Results: In ex vivo sections of intestine muscle cells or isolatedcells, levels of phosphorylated Jak1 and Jak2 were increased compared to those in the normalmargin in the same patient. Treatment of cultured muscle cells isolated from normal marginwith IL-6 elicited the same pattern of increased STAT3(S727) and decreased STAT3(Y705)phosphorylation that was seen in muscle cells in strictured intestine. Tofacitinib selectivelyinhibited IL-6-induced phosphorylation of Jak1 but not Jak2. In IL-6 treated cells, the highlevels of STAT3(S727), low levels STAT3(Y705) phosphorylation, and high TGF-β1 andcollagen IαI levels of expression that are characteristic of strictured intestine were restoredto that of normal intestine with low levels of STAT3(S727), high levels of STAT3(Y705)phosphorylation and low TGF-β1 and collagen IαI expression. Conclusions: Tofacitinibselectively inhibits Jak1 activity in human intestinal muscle. The effects of tofacitinib on IL-6-dependent STAT3 activity and increased expression of profibrotic TGF-β1 and collagenIαI suggests tofacitinib may be useful in susceptible patients expressing a Montreal B2fibrostenotic phenotype of disease. Supported by NIH-DK49691
Sa1734
Pregnane X Receptor Agonists Enhance Intestinal Epithelial Wound Healingand Repair of the Intestinal Barrier Following the Induction of ExperimentalColitisJoshua D. Terc, Ashleigh Hansen, Laurie Alston, Simon A. Hirota
The intestinal epithelial barrier plays a key role in the maintenance of homeostasis withinthe gastrointestinal tract. Barrier dysfunction, leading to increased epithelial permeability,is associated with the inflammatory bowel diseases (IBD). It is thought that the increasedpermeability in patients with IBD may be driven by alterations in the epithelial woundhealing response. To this end considerable study has been undertaken to identify signalingpathways that may accelerate intestinal epithelial wound healing and normalize the barrierdysfunction observed in IBD. In the current study we examined the role of the pregnane Xreceptor (PXR) in modulating the intestinal epithelial wound healing response. Mutationsand reduced mucosal expression of the PXR are associated with IBD, and others have reportedthat PXR agonists can dampen intestinal inflammation. Furthermore, stimulation of the PXRhas been associated with increased cell migration and proliferation, two of the key processesinvolved in wound healing Thus, we sought to test the hypothesis that PXR agonists enhanceintestinal epithelial repair. To study wound healing in vitro, Caco-2 intestinal epithelial cellswere seeded in Ibidi wound assay inserts and grown to confluence. Wound closure wasassessed at 12 and 24 hours following treatment with PXR agonists rifaximin, rifampicinand SR12813. To study intestinal epithelial healing in vivo, mice were treated dextransulphate sodium (DSS; 2.5% w/v in drinking water) for 7 days and allowed to recover foran additional 7 days on normal drinking water. During the recovery phase, mice were treatedwith a rodent specific PXR agonist, pregnenolone 16α-carbonitrile (PCN), or vehicle by oralgarage. FITC-dextran flux was used to assess in vivo permeability after the recovery. Inagreement with our hypothesis, stimulation of Caco-2 cells with rifaximin, rifampicin andSR12813, all potent agonists of the PXR, significantly increased wound closure. This effectwas driven by p38 MAP kinase-dependent cell migration, and occurred in the absence ofcell proliferation. Treating mice with PCN, a potent rodent specific PXR agonist, attenuatedthe intestinal barrier dysfunction observed in the DSS model of experimental colitis, aneffect that occurred independent of its known anti-inflammatory effects. Taken together ourdata indicate that the activation of the PXR can enhance intestinal epithelial repair andsuggest that targeting the PXR may help to normalize intestinal barrier dysfunction observedin patients with IBD. Furthermore, our data provide additional insight into the potentialmechanisms through which rifaximin elicits its clinical efficacy in the treatment of IBD.
Sa1735
IL-17A Modulates the TNF-α-Induced Expression of Inflammatory BowelDisease Susceptibility Genes in Intestinal Epithelial CellsMatthias Friedrich, Julia Diegelmann, Stephan Brand
Background: In contrast to anti-TNF-α antibodies, anti-IL-17A antibodies lacked clinicalefficacy in a trial with patients suffering from Crohn's disease (CD). Therefore, we analyzedhow IL-17A modulates the inflammatory response elicited by TNF-α in intestinal epithelialcells (IEC). Methods: Target mRNA levels in IEC and colonic biopsies were assessed by RNAmicroarray and qRT-PCR. Signaling pathways were analyzed using receptor neutralizationand pharmacological inhibitors. Target protein levels were determined by immunoblotting.Results: Microarray analysis demonstrated that IL-17A alone is a weak inducer of geneexpression in IEC (29 regulated transcripts), but significantly affected the TNF-α-inducedexpression of 547 genes, with a strong amplification of proinflammatory chemokines andcytokines (>200-fold increase of CCL20, CXCL1 and CXCL8). Interestingly, IL-17A differen-tially modulated the TNF-α-induced expression of several IBD susceptibility genes in IEC(increase of JAK2 mRNA, decrease of FUT2, ICOSLG, ICAM1, LTB, and SMAD3 mRNA).For ICOSLG and ICAM1 mRNA, we demonstrated that this is dependent on NF-κB signaling.Negative regulation of ICAM-1 by IL-17A was verified on protein level. The biologicalsignificance of these findings is emphasized by inflamed lesions of IBD patients demonstratingincreased ICAM1 (P=0.028) and JAK2 (P=0.002) transcript levels. Conclusions: We demon-strates the modulation of IBD susceptibility gene mRNA as a novel important property ofIL-17A. Given our microarray data demonstrating a low effect of sole IL-17A stimulationon IEC gene expression, this provides an important explanation for the lack of clinicalefficacy of IL-17A neutralization.
S-284AGA Abstracts
Sa1736
Macrophage Subtypes Disrupt the Epithelium via Deregulated Tight Junctionsand Induction of ApoptosisDonata Lissner, Michael Schumann, Arvind Batra, Lea I. Kredel, Anja A. Kuehl, BrittaSiegmund
Background We have shown that pro-inflammatory macrophage subtypes, as found in thelamina propria of patients with Crohns disease (CD), are capable of disrupting the epithelialbarrier. The exact mechanism has not been described yet. Methods Unpolarized macrophages(M0) were isolated from human peripheral blood and subsequently polarized into M1- andM2-macrophages. The effect of these cells types on epithelial resistance and thus integritywas analysed after co-culture with different epithelial cell lines (Caco-2, T-84, HT-29/B6).Tight junction proteins (claudin-1 and -2, JAM-1, ZO-1, E-cadherin) and caspase-3 and -8were analysed by immunostaining and western blot. Results Compared to regulatory M2-macrophages, pro-inflammatory M0- and M1 macrophages lead to a massive decrease inepithelial resistance. Mainly, this is due to deregulation of tight junction proteins: The pore-forming protein claudin-2 is significantly up-regulated in M0- and M1-exposed epithelialcells, whereas E-cadherin tends to be decreased in these conditions and increased in M2-exposed cells. Additionally, increased caspase-3 in M0-treated epithelial cells reveals induc-tion of apoptosis as a further mechanism. Here, elevated amounts of active caspase-8 suggestdeath receptor mediated apoptosis, rather than via mitochondrial regulation. ConclusionThe disrupting effect of pro-inflammatory macrophages, as found in the lamina propria ofpatients with CD, on the epithelial barrier is due to deregulated tight junction proteins andinduction of death receptor mediated apoptosis.
Sa1737
Osteoprotegerin: A Novel Alarmin in Colonic Epithelial Cells?Raghunath Ramanarasimhaiah, Marina L. Fernandez, Anthony Vella, Francisco A.Sylvester
Background: Osteoprotegerin (OPG) blocks osteoclast formation, is involved in metastaticcancer and may have immune regulatory functions. We have reported that fecal OPG issignificantly increased in active colonic inflammatory bowel disease (IBD). We hypothesizedthat colonic epithelial cells synthesize and secrete OPG into the colonic lumen. Aim: Tocharacterize OPG expression in colonic epithelial cells. Methods: We examined the expres-sion of OPG in colonic endoscopic biopsies from children with normal histology and withIBD and in subcellular compartments of the colonic epithelial cell line Caco-2. We usedimmunohistochemistry (IHC), immunofluorescence (IF) paired with confocal microscopy,Western blot (WB) and immunoprecipitation followed by mass spectrometry (Mass Spec)(primary antibody Cat# IMG103A, Imgenex or Cat# ALX-210-945, Enzo), and ELISA (R&D Systems), as appropriate. OPG mRNA expression was examined with quantitative PCR(qPCR). Recombinant human cytokines were obtained from MBL International Corporationor Cell Signaling Technology. Results: We studied 21 biopsies of children with IBD and11 with normal histology (CTRL). We observed expression of OPG in lamina propriamononuclear cells (LPMC), but the strongest OPG expression was in colonic epithelial cells.Signal was observed in LPMC only in the cytoplasm, but colonic epithelial cells had signalboth in the nucleus and cytoplasm in CTRL. In IBD, nuclear OPG was no longer presentin epithelial cells, while strong signal persisted in the cytoplasm. We then studied Caco-2cells as a model for colonic epithelial cells. At baseline, Caco-2 cells secreted OPG into themedium (mean ± SD, 6.2 ± 0.5 ng/mL OPG. Interleukin (IL)-1β (0.3 ng/mL for 24hrs)increased OPG secretion 2-fold (12.4 ± 0.8 ng/mL; p <0.001); OPG mRNA also increased6-fold compared to control (p<0.001). TNF-α (100 ng/mL) similarly increased OPG expres-sion, but IL-4 and IL-18 had no effect. IF showed a "starry sky" pattern of OPG in thecytoplasm and densely in the nucleus. Presence of OPG in the nucleus was confirmed byWB, ELISA and Mass Spec of nuclear extracts. Conclusion: We confirm for the first timethe presence of nuclear OPG in colonic epithelial cells that is mobilized during inflammation,a pattern akin to IL-33. Our observations suggest that OPG may be an alarmin activatedduring inflammation in IBD.
Sa1738
Paracrine Activity of Necrotic Epithelial IL-1α Mediates Non-Immune CellInteractions and Amplification of Intestinal InflammationMelania Scarpa, Tammy M. Sadler, Gail West, Claudio Fiocchi, Eleni Stylianou
Introduction Excessive intestinal epithelial cell death is a recognized feature of InflammatoryBowel Disease (IBD). However, information on damage-associated molecular patterns(DAMPs), the products of necrosis, and on their function in IBD, is limited. We have shownpreviously that interleukin-1 alpha (IL-1α), is a DAMP produced by necrotic intestinalepithelial cells that activates human intestinal fibroblasts (HIF) cytokine production andexacerbates intestinal injury. We aimed to determine whether intestinal epithelial-fibroblastinteractions depend on IL-1α that is i) produced as a DAMP by intestinal epithelial cells,ii) secreted by live epithelial cells in response to proinflammatory or NLRP3 inflammasomestimuli, iii) endogenous to HIF. Materials and Methods Small interfering RNA (siRNA)targeting human IL-1α (ON-TARGET plus SMARTpool, Dharmacon) or a scrambled controlsiRNA were transfected into HT29 or HIF by nucleofection (Amaxa) for 48 hrs. Transfectedor control HIF were incubated with HT29 necrotic cell lysates (NCL, 1/20 dilution in growthmedium) prepared from cells transfected with IL-1α siRNA or control HT29 cells for 24hrs.Supernatants were collected and analyzed for IL-6 and IL-8 by ELISA. The levels of IL-1αand β-actin mRNA were determined by RT-PCR. HT29 or human monocytes were incubatedwith ultrapure LPS (10ng/ml-1μg/ml) followed by incubation for 24hrs with the proinflamma-tory cytokine TNFα (100ng/ml), or with soluble or particulate activators of the NLRP3inflammasome ATP (5mM), or monsodium urate crystals (MSU, 0.1-0.6 ug/ml). Whole celllysates and supernatants were prepared and levels of IL-1α, IL-1β and IL-8 quantified byELISA. Results NC lysates prepared from HT29 cells in which IL-1α was depleted resultedin impaired production of IL-6 and IL-8 by HIF. These cytokine levels were unaffected bytransfection of IL-1α siRNA in HIF and were unchanged by transfection of the siRNA control.
