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Molecular Mechanisms Responsible for the Selective and Low-Grade Induction of Proinflammatory Mediators in Murine Macrophages by Lipopolysaccharide This information is current as of October 2, 2021. Urmila Maitra, Hui Deng, Trevor Glaros, Bianca Baker, Daniel G. S. Capelluto, Zihai Li and Liwu Li J Immunol 2012; 189:1014-1023; Prepublished online 15 June 2012;

doi: 10.4049/jimmunol.1200857 Downloaded from http://www.jimmunol.org/content/189/2/1014

Supplementary http://www.jimmunol.org/content/suppl/2012/06/15/jimmunol.120085 Material 7.DC1 http://www.jimmunol.org/ References This article cites 92 articles, 37 of which you can access for free at: http://www.jimmunol.org/content/189/2/1014.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Molecular Mechanisms Responsible for the Selective and Low-Grade Induction of Proinﬂammatory Mediators in Murine Macrophages by Lipopolysaccharide

Urmila Maitra,*,1 Hui Deng,*,1 Trevor Glaros,*,1 Bianca Baker,* Daniel G. S. Capelluto,* Zihai Li,† and Liwu Li*

Low-dose endotoxemia is prevalent in humans with adverse health conditions, and it correlates with the pathogenesis of chronic inflammatory diseases such as atherosclerosis, diabetes, and neurologic inflammation. However, the underlying molecular mechanisms are poorly understood. In this study, we demonstrate that subclinical low-dose LPS skews macrophages into a mild proinflammatory state, through cell surface TLR4, IL-1R–associated kinase-1, and the Toll-interacting protein. Unlike high-dose LPS, low-dose LPS does not induce robust activation of NF-kB, MAPKs, PI3K, or anti-inflammatory mediators. Instead, low-dose LPS induces activating transcription factor 2 through Toll-interacting protein–mediated generation of mitochondrial reactive oxygen Downloaded from species, allowing mild induction of proinflammatory mediators. Low-dose LPS also suppresses PI3K and related negative regulators of inflammatory genes. Our data reveal novel mechanisms responsible for skewed and persistent low-grade inflammation, a cardinal feature of chronic inflammatory diseases. The Journal of Immunology, 2012, 189: 1014–1023.

lightly elevated levels of circulating bacterial endotoxin level (17–22). As a consequence, strategies targeting low-dose (∼1–100 pg/ml) persists in humans with adverse health endotoxemia and low-grade inflammation may hold significant http://www.jimmunol.org/ S conditions and/or lifestyles such as chronic infections, promise in not only the treatment, but also the prevention or re- obesity, aging, chronic smoking, and drinking (1–7). Compro- versal, of these debilitating diseases. Despite its significance, the mised mucosal barriers, altered commensal microbiota, and vas- molecular mechanism underlying the effect of low-grade endo- culature leakage collectively contribute to the mild and persistent toxemia is neither well studied nor properly understood. elevation of plasma endotoxin in these individuals. In contrast to Host macrophages are the most potent responders to the bacte- high-dose endotoxin that induces a robust yet transient inflam- rial endotoxin LPS (23). Given the fact that a high dose of cir- matory response, “subclinical” low-dose endotoxin causes low- culating LPS (.10 ng/ml) can cause acute septic shock (24–26), grade yet persistent inflammatory responses from the host, as re- almost all available mechanistic studies regarding cellular re- by guest on October 2, 2021 flected in the mildly sustained levels of inflammatory mediators sponses to endotoxin used high dosages of LPS (.10 ng/ml). High (1, 8–13). This may underlie the initiation and propagation of doses of LPS cause a robust induction of various proinflammatory chronic pathological diseases, including cardiovascular diseases, mediators in macrophages through the TLR4 pathway (27). In the diabetes, Parkinson’s disease, and other chronic inflammatory meantime, high-dose LPS is also capable of inducing the ex- syndromes (8, 14–16). Given the prevalence of low-dose endo- pression of anti-inflammatory mediators such as IL-10. This toxemia and low-grade inflammation in humans at various stages serves as a compensatory mechanism for the host to dampen ex- of life, the economic and health tolls are reaching a pandemic cessive inflammation (28, 29). Molecular mechanisms responsible for the effect of LPS were largely obtained in the context of high-dose LPS. Through TLR4 *Department of Biological Sciences, Virginia Polytechnic Institute and State Univer- sity, Blacksburg, VA 24061; and †Department of Microbiology and Immunology, and other coreceptors, high-dose LPS opens up a flood gate of Medical University of South Carolina, Charleston, SC 29425 intracellular pathways that eventually lead to the activation of 1U.M., H.D., and T.G. contributed equally to this work. diverse transcription factors such as NF-kB and AP-1 (30, 31). Received for publication March 19, 2012. Accepted for publication May 16, 2012. Collectively, these transcription factors contribute to the robust This work was supported by grants from the American Heart Association, the Na- induction of proinflammatory mediators (31). The pathway lead- tional Institutes of Health, and funds from Virginia Polytechnic Institute and State ing to the activation of NF-kB is the most extensively studied and University (to L.L.). Z.L. is supported by National Institutes of Health Grant AI070603. is relatively well defined (32, 33). In this classical pathway, TLR4 activates the IL-1R–associated kinases (IRAK)-4, -2, and -1 via T.G., U.M., H.D., and B.B. conducted the experiments; L.L., T.G., U.M., H.D., and B.B. designed the experiment and analyzed the data; Z.L. and D.G.S.C. provided the MyD88 adaptor molecule (32, 33). IRAKs then recruit TNFR- critical reagents and comments; and U.M. and L.L. wrote the manuscript. associated factor (TRAF)6/2 and contribute to the activation of Address correspondence and reprint requests to Prof. Liwu Li, Department of Bio- MAPKKKs (e.g., TAK1, MLK3, and Tpl2), the phosphorylation logical Sciences, Virginia Polytechnic Institute and State University, Life Science 1 of the IkB kinase (IKK)a/b complex leading to the phosphory- Building, Washington Street, Blacksburg, VA 24061. E-mail address: [email protected] lation and degradation of IkB, and the eventual release and nu- The online version of this article contains supplemental material. clear translocation of p65/RelA. The IRAK-1/TRAF complex is Abbreviations used in this article: ATF, activating transcription factor; BMDM, bone marrow-derived macrophage; ChIP, chromatin immunoprecipitation; IKK, IkB ki- also responsible for the activation of MAPKs and downstream nase; IRAK, IL-1R–associated kinase; NAC, N-acetyl cysteine; ROS, reactive oxy- transcription factors such as AP-1. Although the potent activation gen species; Tollip, Toll-interacting protein; TRAF, TNFR-associated factor; WT, of the TLR4 pathway is capable of robustly inducing the ex- wild-type. pression of proinflammatory mediators, it also strongly induces Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 negative regulators at multiple levels, including IkBa (the nega- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200857 The Journal of Immunology 1015 tive regulator of NF-kB), PI3K, MKP-1, as well as the inactivation Anti-lamin B (ab-16048) was purchased from Abcam. Anti-C/EBPd (M- of IRAK-1 (33–39). Of particular note, the PI3K pathway 17), anti–MKP-1 (M-18), anti–IRAK-1 (F-4), anti-p65 (F-6), anti-GAPDH b attenuates the expression of proinflammatory mediators and (FL-335), anti–IRAK-M, anti-GSK3 , and anti-ATF2 (C-19) Abs were from Santa Cruz Biotechnology. Anti-mouse IgG and anti-rabbit IgG HRP- facilitates the expression of anti-inflammatory mediators (40). linked Abs were purchased from Cell Signaling Technology. CREB activation by the PI3K pathway facilitates the expression of Mice and cell culture IL-10 (41), whereas induction of MKP-1 by PI3K contributes to the suppression of proinflammatory mediators (42, 43). This may Wild-type (WT) C57BL/6 mice were purchased from Charles River Lab- 2/2 serve to dampen excessive inflammation and reduce collateral oratories. IRAK-1 mice from C57BL/6 background were provided by Dr. James Thomas from the University of Texas Southwestern Medical damage to the host. School. Tollip2/2 mice from C57BL/6 background were provided by Dr. In contrast, low levels of circulating LPS (∼1–100 pg/ml) in Ju¨rg Tschopp from the University of Lausanne at Switzerland. All mice experimental animals and humans with adverse health conditions were housed and bred at the Virginia Polytechnic Institute and State can induce mild, yet selective expression of host proinflammatory University Animal Facility in compliance with approved Animal Care and mediators (1, 8–13). In well-controlled in vitro studies, low dos- Use Committee protocols of Virginia Polytechnic Institute and State University. Bone marrow-derived macrophages (BMDMs) were isolated ages of LPS (10–100 pg/ml) cause a distinct effect by priming from the tibias and femurs of WT, IRAK-12/2, and Tollip2/2 mice by cells for more robust expression of proinflammatory mediators in flushing the bone marrow with DMEM. The cells were cultured in un- response to a second LPS challenge (44–47). Furthermore, chronic treated tissue culture dishes with 50 ml DMEM containing 30% L929 cell microbial infections or injections of low-level TLR agonists can supernatant. On the third day of culture, the cells were fed with an additional 20 ml fresh medium and cultured for another additional 3 d. Cells exacerbate inflammatory diseases such as atherosclerosis (48, 49). were harvested with PBS, resuspended in DMEM supplemented with 1% Despite emerging recognition of the chronic and pathological FBS, and allowed to rest overnight before further treatments. WT effects of subclinical low-dose endotoxin, no report is available Raw264.7 and GP96 knocked-down (GP96KD) Raw264.7 cells defective Downloaded from regarding the underlying molecular mechanism. To our knowl- in cell surface TLR4 were maintained as described previously (52). edge, we provided the first evidence indicating that subclinical Cell transfection doses of LPS (,100 pg/ml) fail to activate the classical NF-kB MEF cells were cultured in complete DMEM medium, as previously de- pathway and its ensuing negative feedback loops (50). scribed. The endotoxin levels within the culture media were below the The present study aims to reveal the intracellular mechanisms detectable range. Transfections with enhanced GFP-Tollip expression responsible for the mild yet selective expression of proinflamma- plasmid were performed using Lipofectamine 2000 reagent (Invitrogen) http://www.jimmunol.org/ tory mediators induced by low-dose endotoxin. Based on previous according to the manufacturer’s instructions. Twenty-four hours after transfection, the stably transfected cells were selected using G418 anti- observations that IRAK-1 and Toll-interacting protein (Tollip) are biotics (400 mg/ml) for 4 wk before use in subsequent experiments. selectively involved in mediating macrophage responses to low- Confocal microscopy dose endotoxin (50, 51), we examined the molecular networks involving IRAK-1 and Tollip that are responsible for the selective The GFP-Tollip–transfected MEF cells were plated in 35-mm glass bottom induction of proinflammatory mediators by low-dose endotoxin. petri dishes (MatTek). For staining of mitochondria, cells were incubated Our study reveals that mitochondria reactive oxygen species (ROS), with 75 nM MitoTracker Red (Invitrogen) for 20 min at 37˚C in darkness. After washing three times with PBS, cells were fixed with paraformalde- induced by low-dose LPS through IRAK-1 and Tollip, contribute to hyde (4%) in PBS for 15 min at room temperature and then washed three by guest on October 2, 2021 the expression of proinflammatory mediators through activating times with PBS. The nuclei were stained using DAPI. Fluorescence images transcription factor (ATF)2. Furthermore, our data indicate that were obtained with a Zeiss LSM510 laser-scanning confocal microscope. low-dose LPS suppresses PI3K pathway involved in the expres- MitoTracker Red was excited with a 543-nm laser line and its emission was collected between 590 and 640 nm. sion of anti-inflammatory mediators. Analysis of protein and mRNA Materials and Methods Cells were harvested after specified treatments and washed with PBS. The Reagents cells were resuspended in a lysis buffer containing protease inhibitor mixture (Sigma-Aldrich) and subjected to SDS-PAGE. The protein bands LPS (Escherichia coli 0111:B4) was purchased from Sigma-Aldrich. Anti- were transferred to an immunoblot polyvinylidene difluoride membrane IkBa (no. 9242), p-JNK (no. 9251), p-ERK (no. 4370), p-p38 (no. 9211), (Bio-Rad) and subjected to immunoblot analysis with the indicated Abs. and p-ATF2 (#9225) Abs were obtained from Cell Signaling Technology. Total RNA was extracted using an Isol-RNA lysis reagent (Invitrogen) and

FIGURE 1. Low-dose LPS selectively induces mild and prolonged expression of proinflammatory mediators. (A) WT BMDMs were treated with either low-dose LPS (50 pg/ml) or high-dose LPS (100 ng/ ml) for the specified time periods. Total RNA was isolated, and real-time RT-PCR assays were performed to determine the expression levels of proinflammatory mediators such as Il6 and Tnfa.(B)WT BMDMs were treated with either low-dose LPS (50 pg/ml) or high-dose LPS (200 ng/ml) for the specified time periods. The levels of anti-inflammatory Il10 and Ccl22 were measured by real-time RT-PCR. The relative transcript levels were standardized against Gapdh levels. Data were representative of at least three independent experiments. *p , 0.05. 1016 MECHANISMS FOR SKEWED LOW-GRADE INFLAMMATION IN MACROPHAGES cDNA was generated with a high-capacity cDNA reverse transcription kit isolation procedure as per the manufacturer’s instructions. The purity of (Applied Biosystems) followed by analysis using SYBR Green Supermix on the mitochondrial fraction was determined using Western blot analysis for an iQ5 thermocycler (Bio-Rad). The relative levels of different transcripts mitochondrial resident protein cyclooxygenase IV and cytosolic protein, were calculated using the DDCt method, and results were normalized based GAPDH. on the expression of Gapdh within the same experimental setting. The For the detection of mitochondrial ROS production, we used the mi- relative level of mRNA in untreated WT cells was adjusted to 1 and served tochondrial- sensitive dye MitoSOX Red (Invitrogen). BMDMs and MEF as the basal reference value. The following primer sets were used: mouse cells were plated in DMEM without phenol red prior to the start of the Gapdh forward, 59-AAC TTT GGC ATT GTG GAA GGG CTC-39, re- experiment. Cells were then stained with 1 mM Hoescht 34580 (Invitrogen) verse, 59-TGG AAG AGT GGG AGT TGC TGT TGA-39; mouse Il6 for 10 min at 37˚C for normalization of fluorescence data. After washing forward, 59-ATC CAG TTG CCT TCT TGG GAC TGA-39, reverse, 59- three times with 1 ml HBSS (Invitrogen), cells were stained with 5 mM TAA GCC TCC GAC TTG TGA AGT GGT-39; mouse Ccl22 forward, 59- MitoSOX Red for 20 min at 37˚C. Cells were then washed with HBSS as AGG CTC AGC AAG CCC TAT TCT TCT-39, reverse, 59-GAA TGT GTT mentioned above prior to LPS treatment for the specified time periods. TCC CAG CTG CCA CAT-39; mouse Il10 forward, 59-GCT CTT GCA Fluorescent wavelength pairs for the individual dyes were 510/580 nm for CTA CCA AAG CCA CAA-39, reverse, 59-AGT AAG AGC AGG CAG MitoSOX Red and 392/440 nm for Hoescht 34580 on a Spectromax M2e CAT AGC AGT-39; mouse Tnfa forward, 59-AGC CGA TGG GTT GTA plate reader (Molecular Devices). CCT TGT CTA-39, reverse, 59-TGA GAT AGC AAA TCG GCT GAC GGT-39. Chromatin immunoprecipitation analysis Analysis of mitochondrial proteins and ROS BMDMs were cross-linked using 1% formaldehyde solution in complete media for 15 min with gentle rocking every 3 min at room temperature. Mitochondrial protein fractions were prepared using a mitochondria iso- Cells were washed twice with cold PBS and treated with a glycine solution lation kit from Thermo Scientific. Briefly, BMDMs were grown on 150- for 5 min to stop the cross-linking reaction. Cells were then lysed in an ice- mm2 tissue culture plates and treated with LPS as indicated prior to the cold buffer containing SDS and protease inhibitor mixture. The samples Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Low-dose LPS does not induce the classical NF-kB and MAPK pathways, nor negative suppressors for inflammatory responses. (A)Low- dose LPS fails to induce IkBa degradation. WT BMDMs were treated with either low-dose LPS (50 pg/ml) or high-dose LPS (100 ng/ml) for the indicated times. The levels of IkBa were determined by Western blot. The same blots were probed with Abs specific for GAPDH as loading controls. (B) Low-dose LPS failed to activate the MAPK pathways. Whole-cell lysates from WT cells treated with either low-dose (50 pg/ml) or high-dose (200 ng/ml) LPS were harvested and used to determine the levels of phosphorylated JNK, p38, and ERK. The total levels of JNK, p38, and ERK were used as loading controls. (C) Low-dose LPS fails to induce the negative regulators, including MKP-1. Total levels of MKP-1 were determined by Western blot. (D) Low-dose LPS reduces IRAK-M. Total levels of IRAK-M from cells treated with either low- or high-dose LPS were measured by Western blot. (E) Low-dose LPS fails to degrade IRAK-1. IRAK-1 levels were determined from cells treated with either low- or high-dose LPS by Western blot. Levels of GAPDH were used as loading controls in (C), (D), and (E). (F) PI3K pathway is activated by high-dose LPS and suppressed by low-dose LPS. Whole-cell lysates from cells treated with either low- or high-dose LPS were harvested and used to determine the levels of phosphorylated Akt. The total levels of Akt were used as the loading control. (G) Nuclear GSK3b is induced by low-dose LPS and reduced by high-dose LPS. Nuclear lysates from cells treated with low- or high-dose LPS were harvested and the levels of GSK3b were determined by Western blot. The levels of lamin B were used as a loading control. Panels are representative of three independent experiments. (H) ChIP analysis to detect the binding of RelB to the Il6 promoter in response to high- and low-dose LPS. WT BMDMs were treated with either low- or high-dose LPS for the indicated time periods. The samples were immunoprecipitated using a RelB-specific Ab and analyzed by PCR using primers spanning the promoter region of murine Il6. The Journal of Immunology 1017 were then sonicated in an ice water bath to shear the chromatin for 10 min tion, but rather reduced the residual levels of phosphorylated Akt. (30 s on and 30 s off). The sheared chromatin was processed using the Chip- As a consequence, the nuclear levels of GSK3b were induced by IT Express Kit (Active Motif). The immunoprecipitated chromatin was low-dose LPS and reduced by high-dose LPS (Fig. 2G). analyzed by PCR using primer pairs that span the proximal promoter regions of mouse Il6. The primer sequences used to amplify the enriched chro- Previous reports also indicate that high-dose LPS recruits the matin samples with the mouse Il6 promoter are as follows: forward primer, suppressive RelB to the promoter of inflammatory genes and 59-TCC CAT CAA GAC ATG CTC AAG TGC-39, reverse, 59-AGC AGA accounts for the endotoxin tolerance (55, 56). After an initial 9 ATG AGC TAC AGA CAT CCC-3 . transient decrease in RelB, high-dose LPS is well documented Statistical analysis to induce a late-phase RelB through NF-kB– as well as PI3K- mediated expression (56, 57). However, no report is available Results are expressed as means 6 SD. Statistical significances between groups were determined using a two-tailed Student t test. A p value of regarding the status of RelB in cells challenged with low-dose ,0.05 was considered statistically significant. LPS. To further test the involvement of RelB in differential gene expression induced by low- and high-dose LPS, we per- Results formed chromatin immunoprecipitation (ChIP) analysis on the Mild and selective induction of proinflammatory mediators by promoter of the Il6 gene. As shown in Fig. 2H, high-dose LPS subclinical low-dose LPS (100 ng/ml) caused a transient decrease of suppressive RelB from We first examined the expression profile of various pro- and anti- the Il6 promoter (2 h time point), allowing for transient induction inflammatory genes in macrophages challenged with either low- or of Il6 expression. The RelB levels at the Il6 promoter subse- high-dose LPS. WT BMDMs were treated with low-dose (50 pg/ quently rose significantly at the 4 h time point, corresponding to the development of endotoxin tolerance (56). In stark contrast, ml) or high-dose LPS (100 ng/ml) for the specified time periods. As Downloaded from expected, high-dose LPS robustly induced the message expression low-dose LPS caused prolonged removal of RelB from the Il6 of both proinflammatory (Il6, Tnfa) and anti-inflammatory (Il10, promoter at both time points (Fig. 2H). Ccl22) mediators (Fig. 1). In contrast, low-dose LPS only induced Subclinical-dose LPS preferentially activates ATF2 and mild expression of proinflammatory mediators, but failed to in- C/EBPd duce any noticeable expression of anti-inflammatory mediators AP-1 family proteins are among potential transcription factors (Fig. 1). The levels of proinflammatory Tnfa and Il6 induced by responsible for the mild expression of Tnfa and Il6 . We noticed http://www.jimmunol.org/ high-dose LPS peaked transiently at 4–6 h and dropped signifi- a rapid and significant induction of ATF2 protein by low-dose cantly at 8–10 h. In contrast, the expression of Tnfa and Il6 induced by low-dose LPS was prolonged and persisted throughout the time course. Subclinical-dose LPS fails to induce robust activation of NF-kB and MAPK as well as negative regulators To clarify the underlying molecular mechanisms, we examined the activation status of key TLR4 downstream components, including by guest on October 2, 2021 NF-kB and MAPKs. As shown in Fig. 2A, high-dose LPS induced a rapid degradation of IkB, an indication of the activation of the classical NF-kB pathway. In contrast, low-dose LPS failed to induce noticeable degradation of IkB. High-dose LPS also triggered robust activation of MAPKs, including p38, ERK, and JNK (Fig. 2B). In contrast, low-dose LPS failed to induce noticeable activation of all three MAPKs. As well noticed in the field, high-dose LPS also induces negative regulators, including MKP-1 and IRAK-M, that serve to dampen the expression of proinflammatory mediators (43, 53). We then examined the expression of MKP-1 and IRAK-M by high- versus low-dose LPS. As shown in Fig. 2C, high-dose LPS induced significant expression of MKP-1. In contrast, low-dose LPS failed to induce MKP-1. Intriguingly, low-dose LPS reduced the levels of IRAK-M (Fig. 2D). Other mechanisms of compensatory suppression/tolerance triggered by high-dose LPS include degra- FIGURE 3. Low-dose LPS selectively induces ATF2. (A) Low-dose dation of IRAK-1 (33). As shown in Fig. 2E, high-dose LPS led LPS induces a rapid increase in ATF2 protein levels in WT BMDMs. to rapid modification and degradation of IRAK-1, as reflected in WT cells were treated with low-dose (50 pg/ml) or high-dose (100 ng/ml) the occurrence of an upper band typical of ubiquitinated IRAK-1, LPS for the indicated times. Total ATF2 protein levels were determined by and later disappearance of IRAK-1. In contrast, low-dose LPS Western blot, and GAPDH levels were used as loading controls. (B)Low- failed to induce IRAK-1 ubiquitination and degradation. dose LPS recruits ATF2 to the proximal promoter of the proinflammatory Activation of the PI3K pathway by high-dose LPS was shown to gene IL-6 in WT BMDMs. BMDMs were either untreated or treated with be responsible for the induction of negative regulators, including 50 pg/ml LPS for 2 h and then subjected to ChIP assay using an Ab specific MKP-1 and degradation of IRAK-1 (42, 54). Additionally, PI3K to ATF2 and primers specific to the proximal promoter of IL-6. The same samples were immunoprecipitated using IgG as a nonspecific control, and activation also leads to the expression of anti-inflammatory medi- input DNA was analyzed as the loading control. Arrow points to the ators such as IL-10 (40, 41). Thus, we evaluated the activation specific amplification product. *Nonspecific band. (C) Low-dose LPS status of the PI3K pathway in cells treated with either low- or induces a late C/ebpd expression in WT BMDMs. WT cells were treated high-dose LPS. As shown in Fig. 2F, high-dose LPS led to robust with either low-dose (50 pg/ml) or high-dose (100 ng/ml) LPS for the phosphorylation of Akt, an indication of PI3K activation. Strik- indicated times. C/ebpd message levels were analyzed by real-time RT- ingly, low-dose LPS not only failed to induce Akt phosphoryla- PCR (n = 3). *p , 0.05. 1018 MECHANISMS FOR SKEWED LOW-GRADE INFLAMMATION IN MACROPHAGES

LPS. The protein levels of ATF2 were similarly and significantly IRAK-1 and Tollip using macrophages harvested from IRAK- induced after 30 min stimulation with high-dose LPS (Fig. 3A). To 1– and Tollip-deficient mice. This is based on previous reports determine whether the induction of ATF2 may be due to its gene suggesting that IRAK-1 and Tollip are involved in mediating the expression, we measured mRNA levels of Atf2 and did not detect effect of low-dose LPS (50, 51). Inconsistent with previous any noticeable increase in Atf2 message within 2 h LPS stimula- reports, the induction of Il6 and Tnfa by low-dose LPS was sig- tion (data not shown). This suggests that LPS may selectively nificantly reduced in IRAK-1– and Tollip-deficient cells (Fig. 4A). stabilize ATF2 protein through a novel mechanism independent of We further examined the protein levels of ATF2 in various cells gene transcription. treated with low-dose LPS. As shown in Fig. 4B, ATF2 protein To confirm that ATF2 contributes to the expression of Il6 in- levels were induced in WT cells treated with low-dose LPS. In duced by low-dose LPS, we performed ChIP analyses to examine contrast, low-dose LPS failed to increase ATF2 protein levels in the recruitment of ATF2 to the Il6 promoter. As shown in Fig. 3B, either IRAK-1– or Tollip-deficient cells. Alternatively, the late low-dose LPS challenge led to significant recruitment of ATF2 to induction of C/ebpd by low-dose LPS is only dependent on IRAK- the proximal promoter of Il6. 1, but not Tollip (Fig. 4C). We and others also reported that a late-phase C/EBPd induction Employing ChIP assays, we confirmed that low-dose LPS failed mediated by IKKε contributes to prolonged expression of proin- to recruit ATF2 to the Il6 promoter in either IRAK-1– or Tollip- flammatory mediators such as Il6 (50, 58). To further confirm this deficient mice (Fig. 4D). In terms of C/EBPd, although its message phenomenon, we measured the levels of C/ebpd in cells treated/ induction is dependent on IRAK-1 instead of Tollip, its active stimulated with low-dose LPS. As shown in Fig. 3C, a late in- recruitment to the Il6 promoter depend on both IRAK-1 and duction of C/ebpd (4–8 h) was observed following low-dose LPS Tollip. LPS-induced recruitment of C/EBPd is reduced in both stimulation. In contrast, high-dose LPS led to a transient induction IRAK-1– and Tollip-deficient cells. Downloaded from of C/ebpd. Tollip-mediated ROS generation in mitochondria IRAK-1 and Tollip are involved in the induction of ATF2 Tollip is a novel TLR intracellular adaptor molecule with poorly To further determine the upstream signaling pathways responsible defined function (59). It contains a lipid-binding C2 domain and an for the mild expression of Il6 and Tnfa, we examined the role of ubiquitin-binding CUE domain (60). Limited studies suggest that http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Low-dose LPS-mediated activation of ATF2 requires IRAK-1 and Tollip. (A) Reduced induction of Il6 and Tnfa in IRAK-1– and Tollip- deficient BMDMs. WT and IRAK-1– and Tollip-deficient BMDMs were treated with low-dose LPS (50 pg/ml) for 4 h and the transcript levels of Il6 and Tnfa were analyzed by real-time RT-PCR. *p , 0.05. (B) Induction of ATF2 in response to low-dose LPS depends on IRAK-1 and Tollip. Whole-cell lysates were prepared from WT and IRAK-1– and Tollip-deficient BMDMs treated with low-dose LPS for the indicated time points and analyzed by Western blot. The same blots were probed with GAPDH as loading controls. The adjusted resting levels of ATF2 in each cell type were set as 1, and the ATF2 levels in cells treated with LPS were compared and plotted (n = 3). *p , 0.05. (C) Low-dose LPS-mediated induction of C/ebpd message is dependent on IRAK-1. WT and IRAK-1– and Tollip-deficient BMDMs were treated with low-dose LPS (50 pg/ml) for 6 h and the C/ebpd message levels were analyzed and plotted as shown. *p , 0.05. (D) Tollip and IRAK-1 are required for low-dose LPS-mediated recruitment of ATF2 to the promoter of Il6.WT and IRAK-1– and Tollip-deficient BMDMs were either untreated or treated with 50 pg/ml LPS for 2 h and subjected to ChIP assay using Abs specific to ATF2 or C/EBPd and primers specific to the proximal promoter of Il6. The same samples were immunoprecipitated using IgG as specificity control and input DNA was analyzed as the loading control. Arrow points to the specific amplification product. *Nonspecific band. The Journal of Immunology 1019

Tollip might be involved in modulation of intracellular traffic (61, significantly reduced the expression of Il6 induced by low-dose 62). To reveal the molecular mechanism responsible for Tollip- LPS (Fig. 6B). As C/EBPd is reported to be induced by IRAK-1 mediated activation of ATF2, we first examined cellular distribu- through IKKε (50, 58), we demonstrated that joint inhibition of tion of Tollip following LPS challenge. First, we fractionated mitochondrial ROS and IKKε completely ablated the induction of cellular lysates from WT BMDMs into soluble and mitochondrial IL-6 by low-dose LPS (Fig 6B). Taken together, our data provide fractions. Western blot analyses revealed that low-dose LPS a mechanism responsible for low-grade inflammation induced by stimulation led to increased Tollip levels in the mitochondrial low-dose LPS (Fig. 7). fraction (Fig. 5A). We realize that subcellular fractionation may contain potential contamination from other membrane sources. Thus, we also employed a confocal microscopy approach to fur- Discussion ther confirm the mitochondria translocation of Tollip. To achieve Our study reveals novel intracellular mechanisms used by sub- this, we generated primary murine embryonic fibroblasts stably clinical low-dose LPS in the skewed and low-grade induction of transfected with enhanced GFP-Tollip fusion protein. Through proinflammatory mediators. First, low-dose LPS fails to activate k confocal microscopy, we detected that low-dose LPS induces either the classical NF- B pathway or pathways activating MAPKs. Tollip translocation to mitochondria (Fig. 5B). Second, low-dose LPS suppresses, instead of activating, the com- Recent studies indicate that mitochondria play a key role in TLR pensatory and anti-inflammatory PI3K pathway. Third, low-dose signaling processes (63, 64). ROS generated from mitochondria LPS utilizes IRAK-1 and Tollip to induce mitochondrial ROS, induced by LPS plays a key role in the activation of the TLR which subsequently activates ATF2. The lack of negative feedbacks downstream pathway. It is particularly relevant that mitochondrial in cells challenged with low-dose LPS may enable prolonged expres- ROS was shown to contribute to the stabilization of transcription sion of proinflammatory mediators, a potential culprit for chronic Downloaded from factors, including HIF1a and ATF2 (65, 66). To test whether inflammatory diseases often associated with low-grade endotox- Tollip is involved in the generation of mitochondrial ROS, we emia. measured the levels of mitochondrial ROS using MitoSOX These mechanisms reconcile recent clinical findings that low- Red, a selective dye for mitochondrial ROS. As shown in Fig. 5C, dose endotoxemia closely correlates with low-grade inflam- low-dose LPS induced mitochondrial ROS in WT cells but failed mation and the pathogenesis of chronic human diseases, including to do so in either IRAK-1– or Tollip-deficient cells. atherosclerosis, diabetes, and neurologic diseases (67–70). Cardinal http://www.jimmunol.org/ features of chronic low-grade inflammation include mild and Inhibition of mitochondrial ROS reduces low-grade induction persistent expression of inflammatory mediators as well as skewed of proinflammatory mediators proinflammatory profiles. For example, aged individuals tend to We further tested the role of ROS in the induction of IL-6 by low- have elevated plasma levels of proinflammatory mediators in- dose LPS by applying a selective ROS inhibitor, N-acetyl cysteine cluding CRP and IL-6 (71, 72), reduced Ag-presentation capa- (NAC). As shown in Fig. 6A, NAC effectively blocked the in- bilities (73, 74), as well as reduced levels of anti-inflammatory duction of ATF2 by low-dose LPS (Fig. 6A). Furthermore, NAC TGF-b and IL-10 (75). Our finding that low-dose LPS fails to by guest on October 2, 2021

FIGURE 5. Low-dose LPS translocates Tollip to the mitochondria and induces ROS. (A) Mitochon- drial localization of Tollip in response to low-dose LPS using subcellular fractionation. WT BMDMs were treated with low-dose LPS and mitochondrial protein fractions were prepared. The purity of the mitochondrial fraction was determined using West- ern blot analysis for mitochondrial-resident protein cyclooxygenase IV and cytosolic protein, GAPDH. (B) Mitochondrial localization of Tollip in response to low-dose LPS using confocal microscopy. The GFP-Tollip–transfected MEF cells were stained with MitoTracker Red to stain the mitochondria. The nuclei were stained using DAPI (blue). The cells were visualized under a laser-scanning confocal microscope (original magnification 3400). The merged images were magnified and shown on the right panel and the colocalization is indicated with an arrow. (C) Involvement of Tollip in mitochondrial ROS generation. WT and IRAK-1– and Tollip-deficient BMDMs were stained with mitochondrial ROS selective dye, MitoSOX Red. Cells were then washed with HBSS and treated with low-dose LPS for 60 min. The cells were also stained with 1 mM Hoescht 34580 for normalization of fluorescent data. Fluorescent intensities were measured and quantified (n = 3). *p , 0.05. 1020 MECHANISMS FOR SKEWED LOW-GRADE INFLAMMATION IN MACROPHAGES

FIGURE 6. Mitochondrial ROS contributes to induction of low-grade inﬂammation. (A) The induction of ATF2 by low-dose LPS is blocked by ROS inhibitor. WT BMDMs were treated with low-dose LPS (50 pg/ ml) in the presence or absence of NAC for the indicated time points. Total cellular lysates were analyzed for ATF2 induction in response to low-dose LPS. The band intensities were quantitated and the relative fold expressions were plotted (n = 3). (B) Inhibition of low- dose LPS-induced Il6 by ROS inhibitor. WT BMDMs were pretreated with DMSO, NAC, or an IKKε inhibitor (IKKin) for 30 min and then treated with low- dose LPS (50 pg/ml) for 4 h. The cells were harvested and the total RNA was used to detect the transcript levels of Il6.*p , 0.05. Downloaded from

induce the PI3K pathway and other negative regulators of in- 50 pg/ml LPS, a much lower LPS concentration (∼5 pg/ml) is able flammatory pathways (MKP-1, SOCS1, and degradation of IRAK- to maintain the expression of IL-6. We realize that human plasma 1) provides a potential explanation for skewed proinflammatory environments in vivo as well as cell culture medium in vitro in- profiles associated with low-grade inflammation. When faced with clude a plethora of reagents and stimulants. The exact LPS con- http://www.jimmunol.org/ a higher dose LPS, multiple pathways in macrophages are acti- centration that may elicit a digital activation of macrophages vated that lead to robust induction of both pro- and anti-inflamma- would most likely vary under different conditions. Nevertheless, tory mediators. As a consequence, the coordinated and balanced our experiments with precisely added LPS in vitro reveal a fun- portfolio of pro- and anti-inflammatory pathways ensures transient damental principle for the novel nature of macrophage activation. inflammatory responses and subsequent resolution. Perhaps the Our data reinforce the notion that mitochondrial ROS plays a levels of endotoxin during low-grade inflammation may fall below key role in innate immunity regulation by LPS. Recent studies the “threshold of alarm” that can effectively invoke an anti-inflammatory resolution, thus undermining the long-term danger of chronic by guest on October 2, 2021 inflammatory diseases (Fig. 7). The lack of robust NF-kB activation and limited activation of ATF2 and C/EBPd identified in this study may underlie the mild expression pattern of proinflammatory mediators. Although ATF2 and C/EBPd may not be capable of robustly inducing the expression of proinflammatory mediators, they are also not capable of evoking negative feedbacks. Instead, both ATF2 and C/EBPd have been shown to induce a positive feedback that leads to their own expression (30, 76). The selective presence of a positive feedback implies a novel “digital” or “bi-stable” toggle switch controlling the activation of macrophages by low-dose endotoxin. In contrast to a linear or “analog” switch due to the activation of a linear pathway, a digital switch owing to a positive feedback circuit can build up an “energy reservoir” and only be turned on when the concentration of stimulant reaches a critical threshold (77–79). Alternatively, once the cells are activated, the presence of the energy reservoir would allow the cells to stay activated with significantly less stimulant. This phenomenon, called “hysteresis,” is well documented in relevant biological contexts, such as cell cycle and DNA annealing (77–79). This hysteresis, which has not been applied in leukocyte biology, would nicely explain several key features of leukocyte physiology and pathology. For example, our immune response is built to withstand challenges without FIGURE 7. A schematic illustration of potential mechanisms responsi- easily getting inflammatory diseases. However, once humans are ble for low-grade inflammation induced by low-dose LPS. Low-dose LPS activates ATF2 through IRAK-1 and Tollip-mediated production of mito- challenged with a sufficient amount of LPS, due to chronic in- chondrial ROS. In the meantime, low-dose LPS suppresses PI3K, IRAK- fection, obesity, or other inflammatory conditions, the inflamma- M, and other related negative regulators. As a consequence, low-dose LPS tory state will be easy to maintain and difficult to get rid of. induces mild, prolonged, and skewed expression of proinflammatory Indeed, we collected data indicating the critical LPS concentration mediators. In contrast, high-dose LPS potently activated NF-kB pathway, required for inducing IL-6 expression in macrophages is ∼25–50 as well as the PI3K pathway, and causes robust yet transient expression of pg/ml (Supplemental Figs. 1–3). Once the cells are pretreated with pro- and anti-inflammatory mediators. The Journal of Immunology 1021 indicate that mitochondrial ROS induced by LPS participate in the promoter in tolerant macrophages challenged with high-dose LPS. expression of proinflammatory mediators (64, 80). Mechanisti- This mechanism underlies the skewed and low-grade inflamma- cally, LPS is shown to induce the translocation of TRAF6 to tion elicited by low-dose LPS. mitochondria, ubiquitination of ECSIT, and disruption of the Although it is clear that low- and high-dose LPS induces distinct electron transport system (63, 64). These events may collectively downstream signaling networks, the responsible mechanisms at the lead to the generation of mitochondrial ROS. Our data reveal that receptor level are not well understood. TLR4 traffics between en- IRAK-1 and Tollip are key signaling molecules responsible for the dosome and plasma membrane, and it can mediate LPS signaling at generation of mitochondrial ROS. To our knowledge, Tollip is the both locations (89). Our data indicate that the cell surface TLR4 is first TLR4 intracellular adaptor molecule shown to translocate into essential for the effect of low-dose LPS (Supplemental Fig. 4). mitochondria following LPS challenge. Note that Tollip is a novel Given the fact that TLR4 may heterodimerize with multiple other TLR adaptor molecule capable of binding to phosphoinositides, receptors, including CD36 and MAC1 (90–92), we hypothesize a phenomenon first reported by our group (81). Additionally, that low-dose LPS may engage a unique set of receptor configu- Tollip contains an ubiquitin-binding CUE domain that can po- ration at the cell surface, and it may prevent further receptor in- tentially facilitate protein ubiquitination and degradation (82). ternalization. Future studies are needed to determine the receptor Based on our data, future studies are clearly warranted to deter- configuration and related proximal signaling network responsible mine the role and mechanism of Tollip-meditated generation of for mediating the opposing effects of low- versus high-dose LPS. mitochondrial ROS. Importantly, note that the role and regulation In summary, our study reveals novel mechanisms responsible for of Tollip are highly complex. Despite its proinflammatory role in low-grade inflammation induced by low-dose LPS. Key features cells stimulated with low-dose LPS, Tollip may dampen proin- identified include IRAK-1– and Tollip-mediated generation of flammatory responses in cells when the NF-kB pathway is acti- mitochondrial ROS as well as selective suppression of PI3K. The Downloaded from vated (83). Although the mechanisms are far from clear, we opposing effects of low- versus high-dose LPS identified in this hypothesize that Tollip may distribute to distinct subcellular study explain the intriguing paradigm of endotoxin priming and compartments and form distinct protein complexes in cells treated tolerance, and provoke future studies paying close attention to the with varying dosages of TLR agonists. dosages as well as history of LPS challenges experienced by the Our study reveals an intriguing paradigm of PI3K modulation host. Intervention of the unique cellular network activated by low- by low- versus high-dose LPS. Although high-dose LPS is known dose LPS may hold significance promise in designing effective http://www.jimmunol.org/ to activate PI3K, to our knowledge, this is the first study to reveal therapies for low-grade and chronic inflammatory diseases. that low-dose LPS potently suppresses basal PI3K activation. The opposing effects of low- versus high-dose LPS on PI3K may be Acknowledgments responsible for multiple unexplained paradigms. In addition to the We thank Matt Morris for assistance in the harvest and culture of macro- skewed proinflammatory profiles in cells challenged with low- phages and members of the Li Laboratory for discussions. dose LPS, macrophages/monocytes can be programmed to ex- hibit either “LPS tolerance” or “LPS priming” dependent on the Disclosures initial dosage of LPS challenge. High-dose LPS causes tolerance The authors have no conflicts of interest. by guest on October 2, 2021 whereas low-dose LPS causes priming (84). The differential modulation of PI3K by different dosages of LPS may contribute to these opposing effects. Further complementing this claim, our References published data indicate that low-dose LPS activates GSK3b, 1. Cani, P. D., R. Bibiloni, C. Knauf, A. Waget, A. M. Neyrinck, N. M. Delzenne, b and R. Burcelin. 2008. 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