Induction Of Inflammatory Genes Research Articles

Abstract 2863: Mechanisms underlying estrogen-induced inflammation in estrogen-deprived breast cancer cells

Abstract Inflammation is critical for estrogen (E2) to undertake apoptosis in E2-deprived breast cancer cells. However, it remains unclear how E2 induces inflammation. Here, we demonstrate that E2 increases lipid metabolism and accumulates unfolded proteins in the endoplasmic reticulum in E2-deprived breast cancer cells. These two major alterations result in different inflammatory responses. E2 rapidly increases lipid metabolism related genes such as fatty acid desaturase 1 (FADS1) and CCAAT/enhancer binding protein beta (CEBPB) to modulate adipose inflammation related factors, e.g. IL6/IL6R after two hours treatment, which regulate the cellular proliferation or differentiation. Simultaneously, accumulation of the unfolded protein activates unfolded protein response (UPR) sensors, protein kinase RNA-like endoplasmic reticulum kinase (PERK) and inositol-requiring protein 1 alpha (IRE1α) within few hours after exposure to E2. Importantly, inhibition of PERK, rather than IRE1α, is able to completely prevent E2 from induction of late event inflammatory genes associated with apoptosis, such as tumor necrosis factor (TNF) superfamily members, BCL2L11 (Bim), and HMOX1,which are activated by E2 after 48 hours. Consistently, nuclear factor-kappa B (NF-κB) is activated when TNFα is maximal after E2 treatment. Further examination revealed that an anti-inflammatory agent, dexamethasone (Dex), activates glucocorticoid receptor (GR) transcriptional activity and blocks E2-induced apoptosis. These effects can be reversed by the knockdown of GR. A notable mechanistic change is that Dex effectively blocks the phosphorylation of PERK and produces equivalent inhibitory effects on the apoptosis-associated inflammatory genes as the PERK inhibitor. However, Dex has an additional potential to activate lipid metabolism and are additive with E2 to elevate FADS1 and adipokine IL6/IL6R. All of these data, for the first time, describe the mechanisms underlying E2-induced inflammation in E2-deprived breast cancer. It provides important information to restrict the clinical use of glucocorticoids, which will undermine the beneficial effects of E2-induced apoptosis in estrogen deprived breast cancer patients. Citation Format: Ping Fan, Fadeke A. Agboke, Amit Kumar Tyagi, V. Craig Jordan. Mechanisms underlying estrogen-induced inflammation in estrogen-deprived breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2863.

The Cannabinoid Receptor 2 Protects Against Alcoholic Liver Disease Via a Macrophage Autophagy-Dependent Pathway.

Kupffer cells, the resident macrophages of the liver, play a major role in the pathogenesis of alcoholic liver disease. We have previously demonstrated that CB2 receptor protects against alcoholic liver disease by inhibiting alcohol-induced inflammation and steatosis via the regulation of Kupffer cell activation. Here, we explored the mechanism underlying these effects and hypothesized that the anti-inflammatory properties of CB2 receptor in Kupffer cells rely on activation of autophagy. For this purpose, mice invalidated for CB2 receptor (CB2Mye−/− mice) or for the autophagy gene ATG5 (ATG5Mye−/− mice) in the myeloid lineage, and their littermate wild-type mice were subjected to chronic-plus-binge ethanol feeding. CB2Mye−/− mice showed exacerbated alcohol-induced pro-inflammatory gene expression and steatosis. Studies in cultured macrophages demonstrated that CB2 receptor activation by JWH-133 stimulated autophagy via a heme oxygenase-1 dependent pathway. Moreover, JWH-133 reduced the induction of inflammatory genes by lipopolysaccharide in wild-type macrophages, but not in ATG5-deficient cells. The CB2 agonist also protected from alcohol-induced liver inflammation and steatosis in wild-type mice, but not in ATG5Mye−/− mice demonstrating that macrophage autophagy mediates the anti-inflammatory and anti-steatogenic effects of CB2 receptor. Altogether these results demonstrate that CB2 receptor activation in macrophages protects from alcohol-induced steatosis by inhibiting hepatic inflammation through an autophagy-dependent pathway.

Inter‐competetion and conversion among TNF‐α‐induced signaling complexes determine the outcomes of TNF‐α signaling

Inflammatory gene induction, apoptosis and necroptosis are major cellular responses of the cells exposed to tumor necrosis factor (TNF)‐α. Here we systematically investigated inter‐relationships among the key signaling molecules that control these cellular processes to uncover the molecular mechanism underlying the inter‐competetion and conversion among TNF‐α induced signaling complexes that elicit multiple alternative outcomes of TNF‐α in a murine system. By taking advantage of quantitative mass spectrometry, we found that the amount of Receptor interacting protein 1 (Rip1) and Tradd recruited to the TNF receptor 1 (TNFR1) complex is the initial determinant factor that governs the cell's decision to undergo cell death or not. Rip1 in complex I downstream of TNFR1 engagement not only initiates the signaling for inflammatory gene expression but also inhibits Tradd‐dependent recruitment of Fadd and caspase‐8 that can lead to rapid apoptosis. When there is no Rip3 or Mlkl in cells, elimination of the ubiquitination of Rip1 leads to formation of complex II, which triggers slower apoptosis than occurs in the absence of Rip1. Complex II can be converted to the necrosome when there are sufficient Rip3 and Mlkl. Both caspase‐8 and Flip can function in the necrosome to suppress necroptosis. Recruitment of Rsk1 to the necrosome is another layer of security to process necroptosis because Rsk1 inactivates caspase‐8 by phosphorylation. Thus, Rip1 is the central controller for cells to have induction of inflammatory genes and/or cell death; the interplay among Rip3, caspase‐8, Flip and Rsk1 determines whether the cells undergo apoptosis or necroptosis.Support or Funding InformationThis work was supported by the National Basic Research Program of China (973 Program; 2015CB553800), the National Scientific and Technological Major Project (2013ZX10002‐002), the National Natural Science Foundation of China (91429301, 31420103910, 31330047 and 31221065), the Hi‐Tech Research and Development Program of China (863 program; 2012AA02A201), the 111 Project (B12001), the Science and Technology Foundation of Xiamen (No. 3502Z20130027), the National Science Foundation of China for Fostering Talents in Basic Research (Grant No.J1310027) and the Open Research Fund of State Key Laboratory of Cellular Stress Biology, Xiamen University.

Aging augments the impact of influenza respiratory tract infection on mobility impairments, muscle-localized inflammation, and muscle atrophy.

Although the influenza virus only infects the respiratory system, myalgias are commonly experienced during infection. In addition to a greater risk of hospitalization and death, older adults are more likely to develop disability following influenza infection; however, this relationship is understudied. We hypothesized that upon challenge with influenza, aging would be associated with functional impairments, as well as upregulation of skeletal muscle inflammatory and atrophy genes. Infected young and aged mice demonstrated decreased mobility and altered gait kinetics. These declines were more prominent in hind limbs and in aged mice. Skeletal muscle expression of genes involved in inflammation, as well as muscle atrophy and proteolysis, increased during influenza infection with an elevated and prolonged peak in aged mice. Infection also decreased expression of positive regulators of muscle mass and myogenesis components to a greater degree in aged mice. Gene expression correlated to influenza-induced body mass loss, although evidence did not support direct muscle infection. Overall, influenza leads to mobility impairments with induction of inflammatory and muscle degradation genes and downregulation of positive regulators of muscle. These effects are augmented and prolonged with aging, providing a molecular link between influenza infection, decreased resilience and increased risk of disability in the elderly.

Severity and Frequency of Proximal Tubule Injury Determines Renal Prognosis.

AKI increases the risk of developing CKD, but the mechanisms linking AKI to CKD remain unclear. Because proximal tubule injury is the mainstay of AKI, we postulated that proximal tubule injury triggers features of CKD. We generated a novel mouse model to induce proximal tubule-specific adjustable injury by inducing the expression of diphtheria toxin (DT) receptor with variable prevalence in proximal tubules. Administration of high-dose DT in mice expressing the DT receptor consistently caused severe proximal tubule-specific injury associated with interstitial fibrosis and reduction of erythropoietin production. Mild proximal tubule injury from a single injection of low-dose DT triggered reversible fibrosis, whereas repeated mild injuries caused sustained interstitial fibrosis, inflammation, glomerulosclerosis, and atubular glomeruli. DT-induced proximal tubule-specific injury also triggered distal tubule injury. Furthermore, injured tubular cells cocultured with fibroblasts stimulated induction of extracellular matrix and inflammatory genes. These results support the existence of proximal-distal tubule crosstalk and crosstalk between tubular cells and fibroblasts. Overall, our data provide evidence that proximal tubule injury triggers several features of CKD and that the severity and frequency of proximal tubule injury determines the progression to CKD.

Disconnect between adipose tissue inflammation and cardiometabolic dysfunction in Ossabaw pigs.

ObjectiveThe Ossabaw pig is emerging as an attractive model of human cardiometabolic disease due to its size and susceptibility to atherosclerosis, among other characteristics. Here we investigated the relationship between adipose tissue inflammation and metabolic dysfunction in this model.MethodsYoung female Ossabaw pigs were fed a western-style high-fat diet (HFD) (n=4) or control low-fat diet (LFD) (n=4) for a period of 9 months and compared for cardiometabolic outcomes and adipose tissue inflammation.ResultsThe HFD-fed “OBESE” pigs were 2.5 times heavier (p<0.001) than LFD-fed “LEAN” pigs and developed severe obesity. HFD-feeding caused pronounced dyslipidemia, hypertension, insulin resistance (systemic and adipose) as well as induction of inflammatory genes, impairments in vasomotor reactivity to insulin and atherosclerosis in the coronary arteries. Remarkably, visceral, subcutaneous and perivascular adipose tissue inflammation (via FACS analysis and RT-PCR) was not increased in OBESE pigs, nor were circulating inflammatory cytokines.ConclusionsThese findings reveal a disconnect between adipose tissue inflammation and cardiometabolic dysfunction induced by western diet feeding in the Ossabaw pig model.

Thrombin Promotes Sustained Signaling and Inflammatory Gene Expression through the CDC25 and Ras-associating Domains of Phospholipase Cϵ

Phospholipase C-epsilon (PLCϵ) plays a critical role in G-protein-coupled receptor-mediated inflammation. In addition to its ability to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, PLCϵ, unlike the other phospholipase C family members, is activated in a sustained manner. We hypothesized that the ability of PLCϵ to function as a guanine nucleotide exchange factor (GEF) for Rap1 supports sustained downstream signaling via feedback of Rap1 to the enzyme Ras-associating (RA2) domain. Using gene deletion and adenoviral rescue, we demonstrate that both the GEF (CDC25 homology domain) and RA2 domains of PLCϵ are required for long term protein kinase D (PKD) activation and subsequent induction of inflammatory genes. PLCϵ localization is largely intracellular and its compartmentalization could contribute to its sustained activation. Here we show that localization of PLCϵ to the Golgi is required for activation of PKD in this compartment as well as for subsequent induction of inflammatory genes. These data provide a molecular mechanism by which PLCϵ mediates sustained signaling and by which astrocytes mediate pathophysiological inflammatory responses.

ID: 123: PLZF restrains inflammation

Inflammation is critical for host defence, but needs to be carefully controlled to avoid chronic disease or fatal responses. We have shown (Sadler et al. PNAS, 2015) that the BTB/POZ, zinc finger transcriptional regulator PLZF restricts the expression of inflammatory gene products. In accord with this, PLZF-deficient animals mount exaggerated inflammatory responses to infectious stimuli. This protective effect of PLZF is mediated via modulation of macrophage activity and there is increased inflammatory gene induction in the absence of PLZF. By analyzing genome-wide histone modifications we have discovered that PLZF establishes basal activity states of early response genes to maintain immune homeostasis and limit damaging inflammation. PLZF stabilizes a co-repressor complex that encompasses histone deacetylase activity to control chromatin. Further investigation of signaling from Toll-like receptors revealed calcium/calmodulin-dependent protein kinase (CaMK2) activates histone acetyltransferase-1 (HAT1), which then acetylates PLZF. Acetylation of PLZF promotes the assembly of a repressor complex incorporating HDAC3 and the NF- κ B p50 subunit that limits the NF- κ B response (Sadler et al. Nature Commun, 2015). Accordingly, diminishing the activity of CaMK2, the expression levels of PLZF or HAT1, or mutating key residues that are covalently modified in PLZF and HAT1, curtails control of the production of inflammatory cytokines. As we have previously shown that PLZF promotes the antiviral response (Xu et al., Immunity 2009), taken together these results suggest a strategy that could fullfil one of the major goals of immune therapy to retain immune resistance to pathogens while curbing damaging inflammation.

Macrophage Expression of Inflammatory Genes in Response to EMCV Infection

The expression and production of type 1 interferon is the classic cellular response to virus infection. In addition to this antiviral response, virus infection also stimulates the production of proinflammatory mediators. In this review, the pathways controlling the induction of inflammatory genes and the roles that these inflammatory mediators contribute to host defense against viral pathogens will be discussed. Specific focus will be on the role of the chemokine receptor CCR5, as a signaling receptor controlling the activation of pathways leading to virus-induced inflammatory gene expression.

Abstract IA9: Vitamin D: Shining light on pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) occurs in the context of an exuberant desmoplastic response that produces a ‘living chimera’ of tumor and activated stromal components. A prominent feature of the stromal response is the transformation of the Vitamin A storing pancreatic stellate cell (PSC) to activated myofibroblasts. We hypothesized that restoration of the quiescent PSC phenotype could disrupt tumor-stroma crosstalk and thus improve response to chemotherapy. Recently, we have shown that the vitamin D receptor (VDR) is expressed at high levels in activated human pancreatic tumor stroma and, in response to ligand, acts as a master transcriptional regulator of PSCs to reprise the quiescent state. In vivo, a synthetic VDR agonist (Cal) induced signal-dependent transcriptional remodeling of tumor stroma, enhancing the anti-tumor effects of gemcitabine by increasing intratumoral concentration to reduce tumor growth. Secreted factors are key drivers of the communication between tumor and stroma. To explore the impact of PSC secreted factors on gene expression in pancreatic cancer, PDAC cell lines were grown in the presence of stromal conditioned media (StCM). Addition of StCM was sufficient to induce expression of thousands of PDAC genes linked to epigenetic reprograming including genome wide histone hyperacetylation. The gene expression profile induced by stromal factors overlap with those previously identified following oncogenic Kras, suggesting functional complementarity between cell-autonomous and micro-environmental cues. As epigenome modification results in changes in gene expression through histone modifications, we tested a small molecule inhibitors epigenetic regulators for the capacity to inhibit gene expression changes downstream of stromal factors. Paracrine induction of pro-growth and inflammatory genes was inhibited by this small molecule, suggesting that acetyl-lysine readers serve as key transducers of stromal inputs to drive alterations in gene expression in pancreatic cancer cells. This work describes a molecular strategy through which transcriptional reprogramming of tumors and tumor stroma by Vitamin D or epigenetic regulators may serve as a therapeutic portal in the treatment of pancreatic cancer. Citation Format: Mara H. Sherman, Ning Ding, Eric A. Collisson, Michael Downes, Ronald M. Evans. Vitamin D: Shining light on pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA9.

Abstract A42: Paracrine regulation of the pancreatic cancer epigenome

Abstract Development of pancreatic ductal adenocarcinoma (PDAC) follows an increasingly well-defined series of genetic events in the pancreatic epithelium. Less well understood are the steps orchestrating the exuberant desmoplasia that accompanies every case of PDAC. This accumulation of stromal components arises from and is maintained by the reciprocal interactions between genetically altered tumor cells and adjacent stroma. Pancreatic stellate cells (PSCs) are myofibroblast-like cells activated by tissue damage and implicated in wound healing. Recent work suggests that PSCs are key primary drivers of the desmoplastic reaction that characterizes the disease, possibly through their established secretory capacity. We wished to explore the paracrine effects of PSC secreted factors on gene expression in pancreatic cancer cells. To investigate the global gene expression changes induced by stromal factors, PDAC cell lines were grown in “a-stromal” or “stromal” conditions. The addition of structural and diffusible PSC-derived factors significantly altered expression of thousands of genes in the cancer genome, suggesting that these factors exert genome-wide effects on the epigenetic state to so broadly affect transcription level. Stromal inputs lead to broad hyperacetylation of histones in the PDAC epigenome, particularly on gene-activating lysines on histone tails. These epigenetic events coincided with induction of genes implicated in anabolic metabolism, the glycosylation pathway, and inflammation. The gene expression changes induced by stromal factors overlap with those previously identified following oncogenic Kras, suggesting functional complementarity between cell-autonomous and microenvironmental cues characteristic of PDAC. As epigenome modification results in changes in gene expression through the functions of readers, we tested a small molecule inhibitor of a family of epigenetic readers for the capacity to inhibit gene expression changes downstream of stromal factors. Paracrine induction of pro-growth and inflammatory genes was inhibited by this small molecule, suggesting that acetyl-lysine readers serve as key transducers of stromal inputs to drive alterations in gene expression in pancreatic cancer cells. Inhibition of acetyl-lysine sensing reduced pancreatic tumor growth and associated inflammation in vivo, with significant reduction in expression of genes implicated in macromolecule biosynthesis and tumor-permissive immune regulation. This work supports further investigation of epigenetic modulators, in combination with targeted drugs or immunotherapeutic approaches, for the treatment of PDAC. Citation Format: Mara H. Sherman, Eric Collisson, Michael Downes, Ronald M. Evans. Paracrine regulation of the pancreatic cancer epigenome. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A42.

Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

IntroductionEfficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs.ResultsThe findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential.ConclusionThe presented isolation method and subsequent cell expansion in platelet lysate supplemented culture medium permits suitable large-scale propagation of PB-ECFC. For optimal use of PB-ECFCs in clinical settings, our data suggest that 15–20 CPDL is the most adequate maturation stage.

Laquinimod regulates inflammatory gene induction in a human model of reactive astrogliosis (S20.005)

Laquinimod regulates inflammatory gene induction in a human model of reactive astrogliosis (S20.005)

International Union of Basic and Clinical Pharmacology. XCVI. Pattern recognition receptors in health and disease.

Since the discovery of Toll, in the fruit fly Drosophila melanogaster, as the first described pattern recognition receptor (PRR) in 1996, many families of these receptors have been discovered and characterized. PRRs play critically important roles in pathogen recognition to initiate innate immune responses that ultimately link to the generation of adaptive immunity. Activation of PRRs leads to the induction of immune and inflammatory genes, including proinflammatory cytokines and chemokines. It is increasingly clear that many PRRs are linked to a range of inflammatory, infectious, immune, and chronic degenerative diseases. Several drugs to modulate PRR activity are already in clinical trials and many more are likely to appear in the near future. Here, we review the different families of mammalian PRRs, the ligands they recognize, the mechanisms of activation, their role in disease, and the potential of targeting these proteins to develop the anti-inflammatory therapeutics of the future.

The aryl hydrocarbon receptor (AhR) mediates resistance to apoptosis induced in breast cancer cells

The aryl hydrocarbon receptor (AhR) is well known as a ligand binding transcription factor regulating various biological effects. Previously we have shown that long-term exposure to estrogen in breast cancer cells caused not only down regulation of estrogen receptor (ER) but also overexpression of AhR. The AhR interacts with several cell signaling pathways associated with induction of tyrosine kinases, cytokines and growth factors which may support the survival roles of AhR escaping from apoptosis elicited by a variety of apoptosis inducing agents in breast cancer. In this study, we studied the anti-apoptotic role of AhR in different breast cancer cells when apoptosis was induced by exposure to UV light and chemotherapeutic agents. Activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in AhR overexpressing breast cancer cells effectively suppressed the apoptotic response induced by UV-irradiation, doxorubicin, lapatinib and paclitaxel. The anti-apoptotic response of TCDD was uniformly antagonized by the treatment with 3′methoxy-4′nitroflavone (MNF), a specific antagonist of AhR. TCDD's survival action of apoptosis was accompanied with the induction of well-known inflammatory genes, such as cyclooxygenase-2 (COX-2) and NF-κB subunit RelB. Moreover, TCDD increased the activity of the immunosuppressive enzyme indoleamine 2, 3-dioxygenase (IDO), which metabolizes tryptophan to kynurenine (Kyn) and mediates tumor immunity. Kyn also acts as an AhR ligand like TCDD, and kyn induced an anti-apoptotic response in breast cancer cells. Accordingly, our present study suggests that AhR plays a pivotal role in the development of breast cancer via the suppression of apoptosis, and provides an idea that the use of AhR antagonists with chemotherapeutic agents may effectively synergize the elimination of breast cancer cells.

Hemolysis Induced Activation of Monocytes Is Followed By Inactivation Via Heme-Induced HO-1 Expression

Introduction: Sickle cell disease (SCD) is characterized by profound erythrocyte hemolysis. Hemolytic events, both chronic and acute, lead to elevated levels of both hemoglobin and heme in patients. Both hemoglobin and heme are toxic in the vasculature, but the relative contribution of each to the overall state of health in patients is unknown. Nonetheless, hemolysis is emerging as a clear contributor to inflammation and coagulation activation. The mechanism through which hemolysis may contribute to either inflammation or coagulation activation is not well defined. The monocyte, however, is the only white blood cell in the bloodstream that is uniquely positioned to influence both coagulation activation and inflammation. Monocytes can express tissue factor (TF) on their surface and, as such, activate the extrinsic pathway of thrombin generation. Furthermore, monocytes are activated in SCD, and likely contribute to the overall inflammatory state of the disease. Thus, we reasoned that monocyte activation is a likely mechanism through which hemolysis, either via free hemoglobin or free heme, could influence both coagulation and inflammation. Another aspect of hemolysis is the persistent induction of heme-oxygenase-1 (HO-1) in the presence of elevated levels of plasma heme. We also reasoned that any interrogation of the effects of hemolysis on monocyte activation would induce the significant expression of this heme-degrading enzyme potentially mitigating long term hemolytic stress.Objectives: Determine the effects of free hemoglobin, heme, and heme-induced HO-1 expression on monocyte inflammation and pro-coagulant function.Methods: Monocyte activation was measured in response to either purified hemoglobins A or S, or heme, and was measured in THP-1 monocytes. Simultaneous analysis of 84 heme-induced inflammatory genes was conducted via qRT-PCR using the Qiagen RT Profiler PCR array. HO-1 was induced by pre-conditioning monocytes overnight in heme (5uM). Monocyte inactivation was measured using this pre-conditioning, followed by an additional treatment with 10uM heme. Monocyte TF expression was determined via flow cytometry analysis. TF specific pro-coagulant activity was determined in a one stage clotting assay. The expression of HO-1 protein in THP-1 cells was analyzed using ELISA.Results: We found, in terms of monocyte function, purified hemoglobins A or S were essentially inert. Free heme at physiological levels in patients with SCD, however, was a powerful monocyte activator. We found that, in naïve cells, heme induced profound TF expression at the cell surface, increased monocyte TF mRNA, and significantly increased the pro-coagulant potential of monocytes in a TF-dependent manner. Furthermore, we noted an acute increase in heme-modulated inflammatory gene expression in monocytes using a global gene expression analysis. Of note, there was a profound induction of inflammatory genes in the TGF-β family by heme, including BMP7. Members of the growth and differentiation family were heme-inducible as well - including GDF1,2,& 3. Expression of interleukins 3, 6, 8, 9, & 25 was all significantly increased in response to an acute heme exposure. All genes responded in a dose dependent manner to heme. There was no heme-dependent induction of TNFα suggesting a non-classical monocytic response to heme stimulation. However, sustained exposure to heme inactivated the monocyte inflammatory gene response. Furthermore, monocyte pre-conditioning in heme rendered the cells resistant to subsequent heme-induced inflammation or pro-coagulant function. This heme induced inactivation of monocyte was coincident with and linked to a robust monocyte expression of HO-1.Conclusions: We report a novel heme-induced activation of monocytes that can be directly inhibited by heme-induced HO-1 expression. Our data therefore provide evidence for the first time of a link between hemolysis and monocyte inflammatory and pro-coagulant function. These data potentially suggest that, in patients, higher levels of baseline hemolysis might be protective against the sudden, acute hemolytic event in SCD. These events often precede acute pain crisis and acute chest syndrome. Further study of monocyte activation and levels of HO-1 in patients during these events is merited. DisclosuresNo relevant conflicts of interest to declare.

Differential control of Mincle-dependent cord factor recognition and macrophage responses by the transcription factors C/EBPβ and HIF1α.

Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, and its synthetic analog Trehalose-6,6-dibehenate (TDB) bind to the C-type lectin receptors macrophage-inducible C-type lectin (Mincle) and Mcl to activate macrophages. Genetically, the transcriptional response to TDB/TDM has been defined to require FcRγ-Syk-Card9 signaling. However, TDB/TDM-triggered kinase activation has not been studied well, and it is largely unknown which transcriptional regulators bring about inflammatory gene expression. In this article, we report that TDB/TDM caused only weak Syk-phosphorylation in resting macrophages, consistent with low basal Mincle expression. However, LPS-priming caused MYD88-dependent upregulation of Mincle, resulting in enhanced TDB/TDM-induced kinase activation and more rapid inflammatory gene expression. TLR-induced Mincle expression partially circumvented the requirement for Mcl in the response to TDB/TDM. To dissect transcriptional responses to TDB/TDM, we mined microarray data and identified early growth response (Egr) family transcription factors as direct Mincle target genes, whereas upregulation of Cebpb and Hif1a required new protein synthesis. Macrophages and dendritic cells lacking C/EBPβ showed nearly complete abrogation of TDB/TDM responsiveness, but also failed to upregulate Mincle. Retroviral rescue of Mincle expression in Cebpb-deficient cells restored induction of Egr1, but not of G-CSF. This pattern of C/EBPβ dependence was also observed after stimulation with the Dectin-1 ligand Curdlan. Inducible expression of hypoxia-inducible factor 1α (HIF1α) also required C/EBPβ. In turn, HIF1α was not required for Mincle expression, kinase activation, and Egr1 or Csf3 expression, but critically contributed to NO production. Taken together, we identify C/EBPβ as central hub in Mincle expression and inflammatory gene induction, whereas HIF1α controls Nos2 expression. C/EBPβ also connects TLR signals to cord factor responsiveness through MYD88-dependent upregulation of Mincle.

Cyclin‐Dependent Kinase 9 Inhibition Protects Cartilage From the Catabolic Effects of Proinflammatory Cytokines

Cyclin-dependent kinase 9 (CDK-9) controls the activation of primary inflammatory response genes. The purpose of this study was to determine whether CDK-9 inhibition protects cartilage from the catabolic effects of proinflammatory cytokines. Human chondrocytes were challenged with different proinflammatory stimuli (interleukin-1β [IL-1β], lipopolysaccharides, and tumor necrosis factor α) in the presence or absence of either the CDK-9 inhibitor flavopiridol or small interfering RNA (siRNA). The expression of messenger RNA (mRNA) for inflammatory mediator genes, catabolic genes, and anabolic genes were determined by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Cartilage explants were incubated for 6 days with IL-1β in the presence or absence of flavopiridol. Cartilage matrix degradation was assessed by the release of glycosaminoglycan (GAG) and cleaved type II collagen (COL2A) peptides. CDK-9 inhibition by flavopiridol or knockdown by siRNA effectively suppressed the induction of mRNA for inducible nitric oxide synthase by all 3 proinflammatory stimuli. Results from NF-κB-targeted PCR array analysis showed that flavopiridol suppressed IL-1β induction of a broad range of inflammatory mediator genes (59 of 67 tested). CDK-9 inhibition also suppressed the induction of catabolic genes (matrix metalloproteinase 1 [MMP-1], MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5), but did not affect the basal expression of anabolic genes (COL2A, aggrecan, and cartilage oligomeric matrix protein) and housekeeping genes. Flavopiridol had no apparent short-term cytotoxicity, as assessed by G6PDH activity. Finally, in IL-1β-treated cartilage explants, flavopiridol reduced the release of the matrix degradation product GAG and cleaved COL2A peptides, but did not affect long-term chondrocyte viability. CDK-9 activity is required for the primary inflammatory response in chondrocytes. Flavopiridol suppresses the induction of inflammatory mediator genes and catabolic genes to protect cartilage from the deleterious effects of proinflammatory cytokines, without affecting cell viability and functions.

Role of JMJD3 and histone modifications in intermittent hypoxia‐induced neuroinflammation (1120.6)

Microglial phenotypic plasticity enables them to sense and respond to a variety of stimuli. While much of this plasticity involves transcriptional gene regulation that is highly susceptible to epigenetic modulation, little is known about how post‐translational histone modifications regulate microglial activities. We have shown that intermittent hypoxia (IH), a hallmark of sleep apnea, induces microglial inflammation in vivo, but mechanisms underlying IH‐induced microglial activation are unknown. The Jumonji gene family of histone demethylases are hypoxia‐sensitive, and JMJD3 is known to regulate macrophage inflammatory processes, but nothing is known about its regulation of microglial activities, or its role in IH‐induced neuroinflammation. Thus, using in vivo and in vitro models we tested the hypothesis that JMJD3 is necessary for IH‐induced microglial inflammation. In vivo, JMJD3 mRNA is upregulated in microglia immunomagnetically isolated from the cortex and hippocampus of IH‐treated animals, with a time course that correlates with inflammatory gene induction. To test the requirement of JMJD3 in IH‐induced microglial gene expression, we exposed microglial cell cultures to IH in the presence and absence of the small molecule JMJD3 inhibitor GSK‐J4 (7.5μM). IH increased JMJD3 mRNA levels which correlated with a decrease in histone H3 lysine 27 (H3K27) trimethylation, the enzymatic target of JMJD3 activity. IH‐induced IL‐6, IL‐1β, iNOS, and COX‐2 pro‐inflammatory gene expression was blocked in the presence of GSK‐J4, suggesting a critical role of JMJD3 and H3K27 demethylation in IH‐induced inflammation. This is the first study to investigate a Jumonji demethylase in the context of IH, and the first to link IH‐induced microglial inflammation to JMJD3 activities.Grant Funding Source: Supported by NIH R01 HL111598 and the Graduate Women in Science Ruth Dickie Fellowship

Jmjd3-mediated epigenetic regulation of inflammatory cytokine gene expression in serum amyloid A-stimulated macrophages

Serum amyloid A (SAA), a major acute-phase protein, has potent cytokine-like activities in isolated phagocytes and synovial fibroblasts. SAA-induced proinflammatory cytokine gene expression requires transcription factors such as NF-κB; however, the associated epigenetic regulatory mechanism remains unclear. Here we report that Jmjd3, a histone H3 lysine 27 (H3K27) demethylase, is highly inducible in SAA-stimulated macrophages and plays an important role in the induction of inflammatory cytokine genes. SAA-induced Jmjd3 expression leads to reduced H3K27 trimethylation. Silencing of Jmjd3 expression significantly inhibited SAA-induced expression of proinflammatory cytokines including IL-23p19, G-CSF and TREM-1, along with up-regulation of H3K27 trimethylation levels on their promoters. Depletion of Jmjd3 expression also attenuated the release of proinflammatory cytokine genes in a peritonitis model and ameliorated neutrophilia in SAA-stimulated mice. Finally, we observed that Jmjd3 is essential for SAA-enhanced macrophage foam cell formation by oxidized LDL. Taken together, these results illustrate a Jmjd3-dependent epigenetic regulatory mechanism for proinflammatory cytokine gene expression in SAA-stimulate macrophages. This mechanism may be subject to therapeutic intervention for sterile inflammation and atherosclerosis.