Abstract 21: Endothelial Epsins Promote LPS-Induced Inflammation During Septic Shock

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Hong Chen
Keyword(s):  
Septic Shock ◽  
Proinflammatory Cytokine ◽  
Cytokine Production ◽  
Therapeutic Potential ◽  
Lethal Dose ◽  
Myeloid Cell ◽  
Mutant Mice ◽  
Proinflammatory Cytokine Production ◽  
Knock Out ◽  
Lps Challenge

Introduction: Sepsis is caused by a deleterious host response to infection, which is primarily responsible for further injury of host tissue and cause of organ dysfunction. Despite significant progress, the pathophysiology of sepsis and the underlying regulatory mechanisms are still not fully understood. We have established that endothelial epsins play a pivotal role in mediating internalization and degradation of Thrombomodulin after LPS challenge. Hypothesis: Given LPS triggers “cytokine storm” that causes hyper-permeability in the endothelium of lungs and excessive inflammation, we assessed the hypothesis that epsins play a role in promoting endothelial permeability and augmenting inflammation. Methods and Results: Using innovative tissue-specific inducible epsins double knock out animal models, we investigated the role for epsins during sepsis. We administered lethal dose of LPS into endothelial-specific inducible epsins mutant mice, myeloid cell-specific epsins mutant mice, and platelet-specific epsins mutant mice (n>10). We uncover a potent protective role for endothelial epsins deficiency against the development of LPS-induced sepsis, whereas deletion of epsins in myeloid cells offers 40% ~ 50% of protection, and loss of epsins in platelets exhibits no protection. We further show that endothelial epsin-deficiency upregulates Thrombomodulin surface protein expression by preventing its internalization and subsequent degradation induced by LPS exposure. Sustained surface Thrombomodulin activity subsequently impaired the heightened Tissue Factor expression and activation that usually occurs in response to LPS. Given LPS challenge mimics chronic inflammatory conditions, we show endothelial epsin-deficiency downregulates LPS-induced proinflammatory cytokine production and suppresses endothelial hyper-permeability in lungs assessed by ELSA and Evans Blue perfusion, respectively. Conclusions: Endothelial epsins depletion inhibits septic shock after LPS challenge by protecting Thrombomodulin against internalization and degradation, blocking proinflammatory cytokine production and inhibiting endothelial leakage in the lungs, highlighting the therapeutic potential for targeting epsins during sepsis.

scholarly journals 862 Dectin-2, a novel target for tumor macrophage reprogramming in cancer immunotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A903-A903
Author(s):  
Justin Kenkel ◽  
Po Ho ◽  
Sameera Kongara ◽  
Karla Henning ◽  
Cindy Kreder ◽  
...  
Keyword(s):  
Proinflammatory Cytokine ◽  
Antigen Processing ◽  
Cytokine Production ◽  
Immune Cell ◽  
Therapeutic Potential ◽  
Costimulatory Molecule ◽  
Mouse Tumor ◽  
Dependent Manner ◽  
Proinflammatory Cytokine Production

BackgroundTumor-associated macrophages (TAMs) are an abundant immune cell population in most cancers that support tumor progression through their immunosuppressive effects. We discovered that TAMs express the pattern recognition receptor Dectin-2 (Clec4n/CLEC6A), an activating C-type lectin receptor (CLR) that binds to high-mannose glycans on fungi and other microbes and induces protective immune responses against infectious disease. Dectin-2 is selectively expressed by myeloid cells, and upon ligation mediates enhanced phagocytosis, antigen processing and presentation, and proinflammatory cytokine production. Given these properties, we evaluated the therapeutic potential of targeting Dectin-2 using naturally derived ligands. We also generated human Dectin-2-targeted agonistic antibodies capable of robustly activating immunosuppressive ”M2” or TAM-like macrophages.MethodsDectin-2 expression was assessed by flow cytometry, immunohistochemistry, and using public databases. Mouse and human monocytes were differentiated into macrophages using recombinant cytokines or tumor-conditioned media, and stimulation was measured following overnight incubation with Dectin-2 ligands or antibodies. Mouse tumor cell lines were implanted into syngeneic hosts and mice were treated with mannan derived from S. cerevisiae via IT or IV administration.ResultsDectin-2 gene expression is minimal in normal human tissues but elevated across many tumor types, including breast, colon, lung, and kidney cancers. Dectin-2 is strongly expressed by macrophages differentiated in vitro and on primary TAMs. The fungal Dectin-2 ligand mannan stimulated proinflammatory cytokine production (e.g. TNFalpha) and costimulatory molecule expression (e.g. CD86) by macrophages in a Dectin-2-dependent manner. Treatment of tumor-bearing mice with mannan mediated tumor regression in multiple syngeneic tumor models, with high rates of tumor clearance in the MB49 bladder cancer model. These effects were Dectin-2 dependent, as efficacy was not observed when a Dectin-2-blocking antibody was co-administered or in knockout mice lacking Dectin-2 signaling components. Furthermore, depletion of either macrophages or T cells impaired efficacy, suggesting that Dectin-2-stimulated TAMs augment anti-tumor T cell responses. Based on these data, we developed novel Dectin-2 targeted agonist antibodies capable of activating human ”M2” or TAM-like macrophages in vitro to produce an array of proinflammatory cytokines and chemokines akin to tumor-destructive ”M1” macrophages.ConclusionsThe data presented demonstrate the therapeutic potential of targeting Dectin-2 using natural ligands or agonistic antibodies as a novel pan-cancer approach for myeloid cell-directed tumor immunotherapy.Ethics ApprovalAll animal studies were performed in accordance with Institutional Animal Care and Use Committee (IACUC)-approved protocols.

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

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