scholarly journals Human Cytomegalovirus MicroRNAs miR-US5-1 and miR-UL112-3p Block Proinflammatory Cytokine Production in Response to NF-κB-Activating Factors through Direct Downregulation of IKKα and IKKβ

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Meaghan H. Hancock ◽  
Lauren M. Hook ◽  
Jennifer Mitchell ◽  
Jay A. Nelson
Keyword(s):  
Human Cytomegalovirus ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Hcmv Infection ◽  
Cytokine Production ◽  
Lytic Infection ◽  
Wild Type Virus ◽  
Proinflammatory Cytokine Production ◽  
Iκb Kinase ◽  
Tnf Α

ABSTRACTEmerging evidence indicates that human cytomegalovirus (HCMV) manipulates host cell signaling pathways using both proteins and noncoding RNAs. Several studies have shown that HCMV induces NF-κB signaling early in infection, resulting in the induction of antiviral proinflammatory cytokines with a subsequent reduction of these cytokines late in infection. The mechanism for late cytokine reduction is unknown. In this study, we show that HCMV microRNAs (miRNAs) miR-US5-1 and miR-UL112-3p target the IκB kinase (IKK) complex components IKKα and IKKβ to limit production of proinflammatory cytokines in response to interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α). Transfection of miR-UL112-3p and miR-US5-1 mimics reduced endogenous IKKα and IKKβ protein levels, and site-directed mutagenesis of the 3′ untranslated regions (UTRs) identified the binding sites for each miRNA. Infection with mutant viruses lacking these miRNAs resulted in increased levels of IKKα and IKKβ proteins, an impaired ability to control NF-κB signaling at late times of lytic infection, and increased production of proinflammatory cytokines compared to wild-type virus in cell types relevant to HCMV infectionin vivo. These phenotypes were rescued by preexpression of miR-US5-1 and miR-UL112-3p in infected cells or by a miR-US5-1/miR-UL112-3p double mutant virus that expresses short hairpin RNAs (shRNAs) targeting IKKα and IKKβ, demonstrating the gene specificity of the miRNAs. These observations describe a mechanism through which HCMV miRNAs expressed late in the infectious cycle downregulate proinflammatory cytokine production to create a cellular proviral environment.IMPORTANCEHuman cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in transplant recipients and causes hearing loss and mental retardation when acquired congenitally. Initial events during HCMV infection result in the activation of NF-κB signaling, which culminates in the production of IL-6, CCL5, and TNF-α. Several viruses have developed mechanisms to block the antiviral effects of these cytokines. We show here that two HCMV miRNAs, miR-US5-1 and miR-UL112-3p, specifically downregulate IKKα and IKKβ signaling factors necessary to propagate NF-κB signaling and subsequent IL-6, CCL5, and TNF-α production. Regulation of these proinflammatory cytokines during lytic infection and during latency is critical to viral survival in the host.

scholarly journals AB0108 IRAK4 INHIBITION SUPPRESSES PROINFLAMMATORY CYTOKINE PRODUCTION FROM HUMAN MACROPHAGES STIMULATED WITH SYNOVIAL FLUID FROM RHEUMATOID ARTHRITIS PATIENTS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1353.2-1353
Author(s):  
A. Yadon ◽  
D. Ruelas ◽  
G. Min-Oo ◽  
J. Taylor ◽  
M. R. Warr
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Signaling Pathways ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Cytokine Production ◽  
Interleukin 1 ◽  
Proinflammatory Cytokine Production ◽  
Human Macrophages ◽  
Percent Suppression

Background:Rheumatoid arthritis (RA) is characterized by chronic, uncontrolled joint inflammation and tissue destruction. Macrophages are thought to be key mediators in both the initiation and perpetuation of this pathology.1,2The RA synovium contains a complex inflammatory milieu that can stimulate macrophage-dependent production of proinflammatory cytokines through multiple signaling pathways.1,2Existing evidence indicates that toll-like receptors (TLRs) and interleukin-1 receptors (IL-1R) along with their agonists, damage-associated molecular patterns (DAMPs) and IL-1β, are highly expressed in RA joints and are important mediators of synovial macrophage activation and proinflammatory cytokine production.1-9IRAK4 (interleukin-1 receptor-associated kinase 4) is a serine/threonine kinase that facilitates TLR and IL-1R signaling in many cell types, including macrophages.10IRAK4 inhibition represents an opportunity to reduce proinflammatory cytokine production in the joints of patients with RA.Objectives:To investigate the effect of a highly selective IRAK4 inhibitor on proinflammatory cytokine production from human macrophages stimulated with synovial fluid from patients with RA.Methods:Primary human monocytes from 2 independent donors were differentiated for 6 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate human monocyte-derived macrophages (hMDMs). hMDMs were then pretreated with an IRAK4 inhibitor for 1 hour and subsequently stimulated for 24 hours with RA synovial fluid from 5 patients. Culture supernatants were then assessed for secretion of proinflammatory cytokines by MesoScale Discovery.Results:RA synovial fluid stimulation of hMDMs resulted in the production of several proinflammatory cytokines, including IL-6, IL-8, and TNFα. Pretreatment of hMDMs with an IRAK4 inhibitor resulted in the dose-dependent inhibition of IL-6, IL-8, and TNFα production, with an average EC50± SD of 27 ± 31, 26 ± 41, and 28 ± 22 nM, respectively. Maximal percent suppression ± SD of IL-6, IL-8, and TNFα were 76 ± 8.8, 73 ± 15, and 77 ± 13, respectively. To evaluate the specific IRAK4-dependent signaling pathways mediating this response, hMDMs were pretreated with inhibitors of TLR4 (TAK242) and IL-1R (IL-1RA) prior to stimulation with RA synovial fluid. Both TAK242 and IL-1RA inhibited proinflammatory cytokine production. For TAK242, maximal percent suppression ± SD of IL-6, IL-8, and TNFα were 39 ± 25, 48 ± 24, and 50 ± 21, respectively. For IL-1RA maximal percent suppression ± SD of IL-6, IL-8, and TNFα were 18 ± 18, 20 ± 23, and 16 ± 18, respectively. The broad range of inhibition across each stimulation highlights the complexity and variability in the signaling pathways mediating proinflammatory cytokine production from hMDMs stimulated with RA synovial fluid, but demonstrates that RA synovial fluid can stimulate proinflammatory cytokine production in hMDMs, at least partly, through IRAK4-dependent pathways.Conclusion:This work demonstrates that IRAK4 inhibition can suppress proinflammatory cytokine production from macrophages stimulated with synovial fluid from patients with RA and supports a potential pathophysiological role for IRAK4 in perpetuating chronic inflammation in RA.References:[1]Smolen JS, et al.Nat Rev Dis Primers.2018;4:18001.[2]Udalova IA, et al.Nat Rev Rheumatol.2016;12(8):472-485.[3]Joosten LAB, et al.Nat Rev Rheumatol.2016;12(6):344-357.[4]Huang QQ, Pope RM.Curr Rheumatol Rep.2009;11(5):357-364.[5]Roh JS, Sohn DH.Immune Netw.2018;18(4):e27.[6]Sacre SM, et al.Am J Pathol.2007;170(2):518-525.[7]Ultaigh SNA, et al.Arthritis Res Ther.2011;13(1):R33.[8]Bottini N, Firestein GS.Nat Rev Rheumatol.2013;9(1):24-33.[9]Firestein GS, McInnes IB.Immunity.2017;46(2):183-196.[10]Janssens S, Beyaert R.Mol Cell.2003;11(2):293-302.Disclosure of Interests:Adam Yadon Employee of: Gilead, Debbie Ruelas Employee of: Gilead, Gundula Min-Oo Employee of: Gilead, James Taylor Employee of: Gilead, Matthew R. Warr Employee of: Gilead

scholarly journals Pulmonary surfactant protein A and surfactant lipids upregulate IRAK-M, a negative regulator of TLR-mediated inflammation in human macrophages

2012 ◽  
Vol 303 (7) ◽  
pp. L608-L616 ◽  
Author(s):  
Huy A. Nguyen ◽  
Murugesan V. S. Rajaram ◽  
Douglas A. Meyer ◽  
Larry S. Schlesinger
Keyword(s):  
Proinflammatory Cytokine ◽  
Cytokine Production ◽  
Protein A ◽  
Surfactant Protein ◽  
Negative Regulator ◽  
Surfactant Protein A ◽  
Proinflammatory Cytokine Production ◽  
Human Macrophages ◽  
Tnf Α

Alveolar macrophages (AMs) are exposed to frequent challenges from inhaled particulates and microbes and function as a first line of defense with a highly regulated immune response because of their unique biology as prototypic alternatively activated macrophages. Lung collectins, particularly surfactant protein A (SP-A), contribute to this activation state by fine-tuning the macrophage inflammatory response. During short-term (10 min–2 h) exposure, SP-A's regulation of human macrophage responses occurs through decreased activity of kinases required for proinflammatory cytokine production. However, AMs are continuously exposed to surfactant, and the biochemical pathways underlying long-term reduction of proinflammatory cytokine activity are not known. We investigated the molecular mechanism(s) underlying SP-A- and surfactant lipid-mediated suppression of proinflammatory cytokine production in response to Toll-like receptor (TLR) 4 (TLR4) activation over longer time periods. We found that exposure of human macrophages to SP-A for 6–24 h upregulates expression of IL-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR-mediated NF-κB activation. Exposure to Survanta, a natural bovine lung extract lacking SP-A, also enhances IRAK-M expression, but at lower magnitude and for a shorter duration than SP-A. Surfactant-mediated upregulation of IRAK-M in macrophages suppresses TLR4-mediated TNF-α and IL-6 production in response to LPS, and IRAK-M knockdown by small interfering RNA reverses this suppression. In contrast to TNF-α and IL-6, the surfactant components upregulate LPS-mediated immunoregulatory IL-10 production, an effect reversed by IRAK-M knockdown. In conclusion, these data identify an important signaling regulator in human macrophages that is used by surfactant to control the long-term alveolar inflammatory response, i.e., enhanced IRAK-M activity.

Abatacept modulates proinflammatory macrophage responses upon cytokine-activated T cell and Toll-like receptor ligand stimulation

2011 ◽  
Vol 71 (1) ◽  
pp. 80-83 ◽  
Author(s):  
M H Wenink ◽  
K C M Santegoets ◽  
A M Platt ◽  
W B van den Berg ◽  
P L C M van Riel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Cytokine Production ◽  
Proinflammatory Cytokine Production ◽  
Toll Like Receptor ◽  
Activated T Cells ◽  
Activated T Cell ◽  
Ligand Stimulation

ObjectivesWe investigated whether Abatacept might reduce proinflammatory cytokine production by macrophages upon contact with cytokine activated T cells and/or stimulation with TLR ligands.MethodsMacrophages and cytokine stimulated T cells (Tck) were added together in the presence of Abatacept or a control Ig, with or without TLR ligands. The production of cytokines was determined by luminex.ResultsAbatacept reduced Tck-induced production of TNFa by macrophages. Tck and TLR ligands synergistically induced the production of proinflammatory cytokines by macrophages, especially IL-12p70. The production of IL-12p70 coincided with the production of IFNg, which were both reduced in the presence of Abatacept.ConclusionsTck induce the production of TNFa by macrophages and facilitate the highly increased production of proinflammatory cytokines in the presence of TLR ligands. Abatacept was shown to potently suppress these pathways suggesting that its role may extend beyond antigen specific T cell mediated effector function.

scholarly journals Toll-Like Receptor 7 Agonist Therapy with Imidazoquinoline Enhances Cancer Cell Death and Increases Lymphocytic Infiltration and Proinflammatory Cytokine Production in Established Tumors of a Renal Cell Carcinoma Mouse Model

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Eric C. Kauffman ◽  
Huixian Liu ◽  
Michael J. Schwartz ◽  
Douglas S. Scherr
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Death ◽  
T Cell ◽  
Tumor Growth ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Cytokine Production ◽  
Serum Levels ◽  
Proinflammatory Cytokine Production ◽  
Toll Like Receptor

Imidazoquinolines are synthetic toll-like receptor 7 and 8 agonists and potent dendritic cell activators with established anticancer activity. Here we test the hypothesis that imidazoquinoline hasin vivoefficacy within established renal cell carcinoma (RCC) tumors. Immunocompetent mice bearing syngeneic RCC xenografts were treated with imidazoquinoline or placebo at two separate time points. Harvested tumors were assayed by TUNEL/caspase-3/Ki67 immunostains to evaluate cell death/apoptosis/proliferation, and CD3/B220/CD45 immunostains to evaluate T-cell lymphocyte/B-cell lymphocyte/pan-leukocyte tumor infiltration. ELISA measurement of tumor and serum levels of proinflammatory cytokines, IL-6 and MCP-1, was performed. A single imidazoquinoline dose significantly decreased RCC tumor growth by 50% and repeat dosing compounded the effect, without observed weight loss or other toxicity. Tumor immunostaining revealed significant increases in cell death and apoptosis without changes in cell proliferation, supporting induction of apoptosis as the primary mechanism of tumor growth suppression. Imidazoquinoline treatment also significantly enhanced peritumoral aggregation and intratumoral infiltration by T-cell lymphocytes, while increasing intratumoral (but not serum) levels of proinflammatory cytokines. In conclusion, imidazoquinoline treatment enhances T-cell lymphocyte infiltration and proinflammatory cytokine production within established mouse RCC tumors, while suppressing tumor growth via induction of cancer cell apoptosis. These findings support a therapeutic role for imidazoquinoline in RCC.

scholarly journals HMGB1 translocation and release mediate cigarette smoke–induced pulmonary inflammation in mice through a TLR4/MyD88-dependent signaling pathway

2017 ◽  
Vol 28 (1) ◽  
pp. 201-209 ◽  
Author(s):  
Yao Cheng ◽  
Dan Wang ◽  
Bin Wang ◽  
Huanan Li ◽  
Junjie Xiong ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Signaling Pathway ◽  
Cigarette Smoke ◽  
Proinflammatory Cytokine ◽  
Cytokine Production ◽  
Pulmonary Inflammation ◽  
High Mobility ◽  
Lung Epithelial Cells ◽  
Proinflammatory Cytokine Production ◽  
Tnf Α

We performed studies to determine the role of high-mobility group box 1 (HMGB1) in cigarette smoke (CS)–induced pulmonary inflammation. After mice were exposed to five cigarettes four times a day for 3 d, toll-like receptor 4 (TLR4) expression and TLR4-mediated signaling were significantly up-regulated, and HMGB1 had translocated from the nucleus to the cytoplasm in lung epithelial cells and then been released into the extracellular lung space. On CS exposure, inflammatory cell recruitment and proinflammatory cytokine production were significantly increased in lung tissue and bronchoalveolar lavage, and these effects depended on the TLR4 signaling pathway. HMGB1 inhibition decreased the CS-induced inflammatory response, whereas treatment with exogenous HMGB1 aggravated the damage and increased the phosphorylation of JNK, p38, and IκBα in the lungs of wild-type mice but not in TLR4-knockout mice. Blockade of TLR4 action or TLR4 knockout significantly inhibited HMGB1-induced proinflammatory cytokine production in mouse tracheal epithelial (MTE) cells and lung tissues. In addition, a MyD88 deficiency inhibited JNK, p38, and IκBα phosphorylation, and this effect was associated with the suppressed production of TNF-α and IL-1β in MTE cells and lung tissues in response to CS stimulation. Thus HMGB1 activates the NF-κB and JNK/p38 pathways through TLR4/MyD88-dependent signaling and induces an inflammatory response in lungs exposed to CS.

Consistency matters: Consistency in the timing and quality of daily interactions between parents and adolescents predicts production of proinflammatory cytokines in youths

2017 ◽  
Vol 30 (2) ◽  
pp. 373-382 ◽  
Author(s):  
Erika M. Manczak ◽  
Adam K. K. Leigh ◽  
Chia-Ping Chin ◽  
Edith Chen
Keyword(s):  
Relationship Quality ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Cytokine Production ◽  
Leisure Activities ◽  
Proinflammatory Cytokine Production ◽  
Parent Child Relationship ◽  
Parent Child

AbstractThe current study examined whether consistency in day-to-day interactions between children and parents related to inflammatory cytokine production in youths. One hundred twenty-three parents recorded the daily quality of interactions and timing of leisure activities with their adolescent children for 2 weeks, and the degree of variability in those ratings was calculated. One year later, the production of proinflammatory cytokines in youths’ blood was measured in response to in vitro exposure to lipopolysaccharide (a bacterial product). The results indicate that greater variability in parent–child relationship quality related to greater stimulated proinflammatory cytokine production in youths, above and beyond overall relationship quality. Greater variability in the timing of parent–child leisure activities also predicted greater stimulated proinflammatory cytokine production in youths, regardless of the frequency of interactions. In sum, consistency in both the affective and temporal aspects of parent–child relationships may contribute to inflammatory processes in youth.

scholarly journals Macrophage-Derived Cell Lines Do Not Express Proinflammatory Cytokines after Exposure to Bacillus anthracis Lethal Toxin

2001 ◽  
Vol 69 (2) ◽  
pp. 1175-1177 ◽  
Author(s):  
James L. Erwin ◽  
Luis M. DaSilva ◽  
Sina Bavari ◽  
Stephen F. Little ◽  
Arthur M. Friedlander ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Bacillus Anthracis ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Messenger Rna ◽  
Cytokine Production ◽  
Lethal Toxin ◽  
Proinflammatory Cytokine Production ◽  
Present Evidence ◽  
Low Levels

ABSTRACT We present evidence that Bacillus anthracis lethal toxin (LT) suppresses rather than induces proinflammatory cytokine production in macrophages. Suppression is observed with extremely low levels of LT and involves inhibition of transcription of cytokine messenger RNA. Thus, LT may contribute to anthrax pathogenesis by suppressing the inflammatory response.

scholarly journals Pentoxifylline Alone or in Combination with Gentamicin or Vancomycin Inhibits Live Microbe-Induced Proinflammatory Cytokine Production in Human Cord Blood and Cord Blood Monocytes In Vitro

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
Esther M. Speer ◽  
Elizabeth Diago-Navarro ◽  
Lukasz S. Ozog ◽  
David J. Dowling ◽  
Wei Hou ◽  
...  
Keyword(s):  
Cord Blood ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Antimicrobial Agents ◽  
Cytokine Production ◽  
Proinflammatory Cytokine Production ◽  
Human Cord Blood ◽  
Content Type ◽  
E Coli

ABSTRACT Neonatal sepsis and its accompanying inflammatory response contribute to substantial morbidity and mortality. Pentoxifylline (PTX), a phosphodiesterase inhibitor which suppresses transcription and production of proinflammatory cytokines, is a candidate adjunctive therapy for newborn sepsis. We hypothesized that PTX decreases live microbe-induced inflammatory cytokine production in newborn blood. Cord blood was stimulated with live microorganisms commonly encountered in newborn sepsis (Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, or Candida albicans) and simultaneously treated with antimicrobial agents (gentamicin, vancomycin, or amphotericin B) and/or clinically relevant concentrations of PTX. Microbial colony counts were enumerated by plating, supernatant cytokines were measured by multiplex assay, intracellular cytokines and signaling molecules were measured by flow cytometry, and mRNA levels were measured by quantitative reverse transcription-PCR. PTX inhibited concentration-dependent E. coli-, S. aureus-, S. epidermidis-, and C. albicans-induced tumor necrosis factor (TNF) and E. coli-induced interleukin-1β (IL-1β) production in whole blood, with greater suppression of proinflammatory cytokines in combination with antimicrobial agents. Likewise, PTX suppressed E. coli-induced monocytic TNF and IL-1β, whereby combined PTX and gentamicin led to significantly greater reduction of TNF and IL-1β. The anti-inflammatory effect of PTX on microbe-induced proinflammatory cytokine production was accompanied by inhibition of TNF mRNA expression and was achieved without suppressing the production of the anti-inflammatory IL-10. Of note, microbial colony counts in newborn blood were not increased by PTX. Our findings demonstrated that PTX inhibited microbe-induced proinflammatory cytokine production, especially when combined with antimicrobial agents, without enhancing microbial proliferation in human cord blood in vitro, thus supporting its utility as candidate adjunctive agent for newborn sepsis.

scholarly journals Corticotropin-Releasing Hormone Augments Proinflammatory Cytokine Production from Macrophages In Vitro and in Lipopolysaccharide-Induced Endotoxin Shock in Mice

2002 ◽  
Vol 70 (11) ◽  
pp. 6068-6074 ◽  
Author(s):  
Sofia Agelaki ◽  
Christos Tsatsanis ◽  
Achille Gravanis ◽  
Andrew N. Margioris
Keyword(s):  
Proinflammatory Cytokine ◽  
Cytokine Production ◽  
Endotoxin Shock ◽  
Proinflammatory Cytokine Production ◽  
Corticotropin Releasing Hormone ◽  
Necrosis Factor Alpha ◽  
Releasing Hormone ◽  
Tnf Α

ABSTRACT Corticotropin-releasing hormone (CRH) exerts an anti-inflammatory effect indirectly, via cortisole production, and a proinflammatory effect directly on immune cells. The aim of the present work was to examine the effect of CRH on macrophage-derived cytokines both in vitro and in vivo. For the in vitro experiments we used two types of macrophages: (i) the RAW264.7 monocyte/macrophage cell line and (ii) thioglycolate-elicited peritoneal macrophages from BALB/c mice. We have found that CRH enhanced lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 production. For the in vivo experiments we have used the LPS-induced endotoxin shock model in BALB/c mice, an established model for systemic inflammation in which macrophages are the major source of the proinflammatory cytokines responsible for the development of the shock. Administration of antalarmin, a synthetic CRH receptor 1 (CRHR1) antagonist, prior to LPS prolonged survival in a statistically significant manner. The effect was more evident at the early stages of endotoxin shock. CRHR1 blockade suppressed LPS-induced elevation of the macrophage-derived cytokines TNF-α, IL-1β, and IL-6, confirming the role of CRH signals in cytokine expression. In conclusion, our data suggest that CRH signals play an early and crucial role in augmenting LPS-induced proinflammatory cytokine production by macrophages. Our data suggest that the diffuse neuroendocrine system via CRH directly affects the immune system at the level of macrophage activation and cytokine production.

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