IFN-Γ Abrogates Endotoxin Tolerance by Facilitating Toll-Like Receptor-Induced Chromatin Remodeling
Total Page：16
File Type：pdf, Size：1020Kb
IFN-γ abrogates endotoxin tolerance by facilitating Toll-like receptor-induced chromatin remodeling Janice Chena and Lionel B. Ivashkiva,b,1 aGraduate Program in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, New York, NY 10021; and bArthritis and Tissue Degeneration Program, Hospital for Special Surgery, New York, NY 10021 Edited by Ruslan Medzhitov, Yale University School of Medicine, New Haven, CT, and approved September 30, 2010 (received for review June 9, 2010) An important mechanism by which IFN-γ primes macrophages for genes important for host defense concomitant with silencing of enhanced innate immune responses is abrogation of feedback in- inflammatory genes that can cause excessive toxicity) (7, 9, 10). hibitory pathways. Accordingly, IFN-γ abrogates endotoxin toler- IFN-γ is a potent endogenous activator of macrophages that ance, a major negative feedback loop that silences expression of augments responses to activating stimuli such as TLR ligands by inflammatory cytokine genes in macrophages previously exposed various mechanisms (11). One important function of IFN-γ is to endotoxin/Toll-like receptor (TLR) ligands. Mechanisms by which reversal of endotoxin tolerance and restoration of inflammatory IFN-γ inhibits endotoxin tolerance have not been elucidated. Here, cytokine production, which has been shown in vitro and in vivo in we show that pretreatment with IFN-γ prevented tolerization of mice and humans (12–16). Importantly, the in vivo biological primary human monocytes and restored TLR4-mediated induction significance of IFN-γ–mediated reversal of endotoxin tolerance of various proinflammatory cytokines, including IL-6 and TNFα. Sur- has been established in human patients. IFN-γ treatment over- prisingly, IFN-γ did not alter proximal TLR4 signaling defects in comes the down-regulation in serum IL-6 and TNFα levels in tolerized monocytes. Instead, IFN-γ blocked tolerance-associated cancer patients exposed to repeated LPS injections given as an- down-regulation of IL6 and TNF transcription, RNA polymerase II titumor therapy and restores monocyte host defense capabilities recruitment, and NF-κB and CCAAT/enhancer-binding protein β in sepsis patients (17, 18). The latter is significant, because it has transcription factor binding to the IL6 and TNF promoters in toler- become increasingly clear that sepsis patients exhibit tolerance- γ IL6 ized monocytes. The mechanism by which IFN- restored expres- related defects in monocyte and macrophage functions, leading to sion was by facilitating TLR4-induced recruitment of chromatin immunosuppression and increased susceptibility to secondary IL6 IL6 remodeling machinery to the promoter and promoting locus infections that represent a leading cause of mortality in this γ accessibility in tolerized monocytes. Our results suggest that IFN- population (19). Despite these numerous observations estab- overcomes endotoxin tolerance by facilitating TLR-induced chroma- lishing the importance of IFN-γ–mediated abrogation of macro- tin remodeling to allow expression of proinflammatory genes. These γ phage tolerance, underlying molecular mechanisms have not results identify a mechanism by which IFN- promotes activation of been previously investigated. In addition, little is known about macrophages and highlight the importance of chromatin remodeling fl mechanisms of tolerance in primary human macrophages, be- and transcriptional control in the regulation of in ammatory cyto- cause most studies have used human cell lines or murine systems. kine production in tolerant and activated macrophages. In this study, we wished to investigate mechanisms by which IFN- γ abrogates endotoxin tolerance. We used primary human mono- inflammation | innate immunity | macrophage cytes to maximize relevance of results for human subjects and compare tolerance mechanisms with those previously described in ndotoxin tolerance is a phenomenon whereby previous ex- mice. Because IFN-γ–mediated augmentation of TLR responses Eposure of cells or organisms to microbial products, such as has conventionally been viewed to occur through positive enhance- Toll-like receptor (TLR) ligands, induces a transient period of ment of TLR signal transduction, we tested the hypothesis that hyporesponsiveness on subsequent endotoxin challenge and is IFN-γ blocks tolerance by overcoming tolerance-associated defects characterized by diminished release of proinflammatory cyto- in TLR signaling. Unexpectedly, IFN-γ did not alter upstream TLR kines, such as IL-6 and TNFα (1, 2). This negative feedback pathway signaling defects in tolerized monocytes. Instead, we mechanism is important for protecting the host against tissue found that IFN-γ inhibited tolerance-mediated down-regulation damage and lethality caused by excessive inflammation. of IL6 and TNF by promoting recruitment of transcription fac- Tolerance results from a complex interplay among many factors tors NF-κB and CCAAT/enhancer-binding protein β (C/EBPβ) involved in multiple levels of TLR signal transduction that alto- and RNA polymerase II (Pol II) to endogenous gene promoters. gether reinforce the refractory state. Most previous work has fo- IFN-γ restored accessibility of the IL6 promoter in tolerized cused on regulation of proximal TLR signaling and has shown that monocytes by facilitating TLR-induced chromatin remodeling. tolerant macrophages exhibit decreased TLR-induced MAPK and These results provide the first insights into mechanisms that block NF-κB activation because of defective assembly of TLR adaptor endotoxin tolerance and highlight the importance of gene-specific proteins and induction of negative regulators of TLR signaling, such regulation in determining the tolerant state of macrophages. as IL-1 receptor-associated kinase M (IRAK-M), SH2-containing inositol phosphatase (SHIP), and MAPK phosphatase 1 (MKP-1) Results (1–6). However, mechanisms that globally suppress TLR signaling IFN-γ Efficiently Blocks Tolerization of IL-6 and TNFα. We first used cannot explain why certain genes are nontolerizable and are in- primary human monocytes to extend previous work showing that duced after TLR stimulation of tolerant cells (7). More recent work has highlighted the importance of gene-specific regulation in tolerant macrophages, which is mediated by modification of chro- Author contributions: J.C. designed research; J.C. performed research; J.C. and L.B.I. matin to silence a subset of TLR-inducible genes. Silencing is ach- analyzed data; and J.C. and L.B.I. wrote the paper. ieved by acquisition of nonpermissive histone modifications and The authors declare no conflict of interest. a block in TLR-induced nucleosome remodeling, resulting in de- This article is a PNAS Direct Submission. creased accessibility of gene loci to transcription factors (7, 8). Such 1To whom correspondence should be addressed. E-mail: [email protected]. fi gene-speci c regulation allows differential expression of subsets This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of TLR-inducible genes (for example, induction of antimicrobial 1073/pnas.1007816107/-/DCSupplemental. 19438–19443 | PNAS | November 9, 2010 | vol. 107 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1007816107 Downloaded by guest on September 30, 2021 IFN-γ can block the development of endotoxin tolerance. Con- IFN-γ Does Not Alter Upstream TLR Signaling Defects in Tolerized trol and IFN-γ–activated human monocytes were tolerized with Monocytes. Because endotoxin tolerance is widely reported to fi increasing doses of LPS for 24 h before being restimulated with be associated with altered TLR signal transduction, we rst asked γ fl a second dose of LPS, and IL-6 and TNFα produced in response whether IFN- restored proin ammatory cytokine production in to LPS rechallenge were measured (Fig. 1A). LPS tolerization tolerized monocytes by restoring upstream signaling events. Control monocytes responded to LPS stimulation with robust doses as low as 10 pg/mL were sufficient to severely blunt the α activation of JNK, ERK, and p38 MAPKs, whereas tolerized ability of control monocytes to produce both IL-6 and TNF monocytes showed diminished activation of MAPKs (Fig. 2A). proteins on LPS rechallenge (Fig. 1B and Fig. S1A). In contrast, Unexpectedly, IFN-γ did not alter the defects in MAPK activation γ fi IFN- pretreatment ef ciently counteracted tolerance-induced in tolerized monocytes (Fig. 2A and Fig. S2 A–C), despite the down-regulation of IL-6 and TNFα. Interestingly, tolerization of ability of these monocytes to produce high levels of IL-6 and IL-6 was readily prevented by IFN-γ, even at high tolerizing TNFα. IFN-γ did not alter the MAPK dependence of these doses of LPS, whereas tolerization of TNFα was more resistant cytokines, because specific inhibitors of MAPKs still diminished to inhibition by IFN-γ at these high LPS doses. These results IL-6 and TNFα production by tolerized monocytes that had been were highly reproducible in experiments with more than 50 in- preactivated with IFN-γ (Fig. S2D), suggesting that low-level fi dependent blood donors; tolerized control monocytes produced MAPK signaling is suf cient to activate cytokine production in γ– κ α 5.9% of the IL-6 and 8.8% of the TNFα produced by their IFN- treated cells. Control monocytes rapidly degraded I B in κ nontolerized counterparts, whereas tolerized monocytes pre- response to LPS stimulation, leading to NF- B p65 phosphory- lation on Ser536 and translocation into the nucleus (Fig. 2B). activated with IFN-γ produced