ToxSci Advance Access originally published online on May 9, 2006
Toxicological Sciences 2006 92(2):445-455; doi:10.1093/toxsci/kfl012
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Toll-Like Receptor Priming Sensitizes Macrophages to Proinflammatory Cytokine Gene Induction by Deoxynivalenol and Other Toxicants
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* Department of Food Science and Human Nutrition, Department of Microbiology and Molecular Genetics, and Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824
1 To whom correspondence should be addressed at Department of Food Science and Human Nutrition, Michigan State University, 234 G. M. Trout Building, East Lansing, MI 48824-1224. Fax: (517) 353-8963. E-mail: pestka{at}msu.edu.
Received February 23, 2006; accepted May 4, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Activation of the innate immune system might predispose a host to toxicant-induced inflammation. In vitro macrophage models were employed to investigate the effects of preexposure to Toll-like receptor (TLR) agonists on induction of proinflammatory cytokine gene expression by the trichothecene mycotoxin deoxynivalenol (DON) and other toxicants. Priming of the murine RAW 264.7 macrophage line or peritoneal murine macrophages with the TLR4 agonist lipopolysaccharide (LPS) at 100 ng/ml for 4, 8, and 16 h significantly increased DON-induced IL-1ß, IL-6, and TNF-
mRNA expression as compared to LPS or DON alone. The minimum LPS concentration for sensitization of both cell types was 1 ng/ml. LPS priming also potentiated IL-1ß mRNA induction by DON in human whole-blood cultures, suggesting the relevance of the murine findings. As observed for LPS, preexposure to TLR agonists including zymosan (TLR2), poly (I:C) (TLR3), flagellin (TLR5), R848 (TLR7/8), and ODN1826 (TLR9) sensitized RAW 267.4 cells to DON-induced proinflammatory gene expression. Amplified proinflammatory mRNA expression was similarly demonstrated in LPS-sensitized RAW 264.7 cells exposed to the microbial toxins satratoxin G, Shiga toxin, and zearalenone as well as the anthropogenic toxicants nickel chloride, triphenyltin, 2,4-dinitrochlorobenzene, and 2,3,7,8-tetrachlorodibenzodioxin. The results suggest that prior TLR activation might render macrophages highly sensitive to subsequent induction of proinflammatory gene expression by xenobiotics with diverse mechanisms of action. Key Words: TLR; deoxynivalenol; LPS; macrophages; xenobiotics.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mammalian host response to invading pathogens, such as bacteria, fungi, and viruses, comprises innate and acquired immune components (Janeway and Medzhitov, 2002
). Innate immunity, the first line of defense, is invoked, in part, via the Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns (PAMPs) (Takeda and Akira, 2005). The most widely studied TLR agonist, endotoxin, is a biologically active component of the gram-negative bacterial cell wall that is composed of protein and the lipopolysaccharide (LPS), with the latter playing the principal role in inflammatory responses (Raetz and Whitfield, 2002). LPS targets mononuclear phagocytes via TLR4, which respond by producing proinflammatory cytokines (e.g., IL-1, TNF-, and IL-6), bioactive lipids (e.g., prostaglandins), and reactive oxygen species (Schletter et al., 1995). Low-dose LPS exposure stimulates both mononuclear phagocyte function and other host responses that result in removal of invading bacteria, while moderate LPS exposure can evoke tissue injury via neutrophil activation and intravascular coagulation (Roth et al., 1997). High-dose LPS exposure initiates a chain of inflammatory events that results in cell death, frank tissue injury, and organ failure.
Human exposure to LPS is common and occurs via infections as well as via translocation of gut microflora and LPS due to inflammatory bowel diseases, gut injury, liver disease, dietary alteration, and alcohol abuse (Roth et al., 1997). Respiratory exposure to LPS exposure also occurs in a variety of occupational and environmental settings (Rylander, 2002). Even though recent studies with rodent models suggest modest inflammatory responses to low-dose LPS may be insufficient to cause overt injury in the unstressed host, such responses might cause frank tissue injury in a host that is coexposed to chemical stressors. There is extensive evidence suggesting that LPS can influence the magnitude of responses to xenobiotics agents in liver, kidney, respiratory tract, and lymphoid tissue (Ganey and Roth, 2001).
Trichothecenes comprise over 180 sesquiterpenoid metabolites elaborated by fungi found in soil, food, and indoor environments (Pestka et al., 2004). The trichothecenes T-2 toxin and deoxynivalenol (DON) occur in cereal grains following Fusarium colonization and have been linked to gastroenteritis and immune dysfunction (Pestka and Smolinski, 2005), whereas the satratoxins and other macrocyclic trichothecenes potentially contribute to respiratory diseases associated with the indoor air mold Stachybotrys (Islam et al., in press). Trichothecenes induce proinflammatory cytokine and chemokine expression in mononuclear phagocytes via a mechanism known as the ribotoxic stress response that involves activation of multiple intracellular signaling cascades (Pestka et al., 2004; Zhou et al., 2003c, 2005). These pathways are similarly observable within a few hours in experimental mice exposed to DON (Zhou et al., 1998, 2003a,b).
The toxicity of the trichothecene mycotoxins is potentiated by LPS, with the immune system being a primary target. In the mouse, exposure to gram-negative pathogens or LPS potentiates T-2 toxin toxicity (Tai and Pestka, 1988a,b; Taylor et al., 1991). Concurrent exposure to LPS greatly magnifies trichothecene-induced cytokine mRNA and protein expression in murine and human macrophage cultures (Chung et al., 2003a,b; Sugita-Konishi and Pestka, 2001; Wong et al., 1998). LPS coexposure also dramatically amplifies DON-induced proinflammatory cytokine expression and apoptosis in murine immune tissues (Islam and Pestka, 2003; Islam et al., 2002; Zhou et al., 1999, 2000). Coexposure to irradiated, nonviable Listeria (Gram positive) and Salmonella (Gram negative) potentiate proinflammatory cytokine responses to DON in the macrophage (Mbandi and Pestka, in press), suggesting that activation of other TLRs in addition to TLR4 might also potentiate trichothecene toxicity.
We have recently determined that LPS pretreatment (i.e., "priming") sensitizes the mouse for an extended time period to induction of proinflammatory cytokine expression and apoptosis by DON (Islam and Pestka, 2006). A single LPS treatment increases susceptibility to DON for as long as 24 h which was reflected by lower DON threshold doses, markedly amplified and prolonged cytokine response, and increased thymocyte apoptosis. These findings suggest that a primary "danger signal" (Matzinger, 2002) might reprogram a host's innate immune system and render it sensitive to secondary signals by a toxicant. We hypothesized that macrophages might be critical targets for reprogramming of the innate immune system to a toxicant-sensitive state. The goal of this research was to test this hypothesis by determining (1) if LPS priming via TLR4 in vitro can sensitize macrophages to DON-induced proinflammatory gene expression, (2) whether other TLR agonists are capable of priming the macrophage response to DON, and (3) whether LPS priming of macrophages enhances their responsiveness to other toxicants known to induce proinflammatory gene expression. The results suggest that priming of macrophages via multiple TLRs increases their sensitivity to induction of inflammatory gene expression by DON and that, in an analogous fashion, LPS priming via TLR4 increases sensitivity to other toxicants with diverse mechanisms of action.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
LPS derived from Salmonella typhimurium (1.5 x 106 EU/mg), DON, nickel chloride (NiCl2), triphenyltin chloride (TPT), 2,4-dinitrochlorobenzene (DNCB), zymosan, poly (I:C), as well as all other chemicals (reagent grade or better) and media components, except where noted, were purchased from Sigma-Aldrich (St Louis, MO). R848 and ODN848 were obtained from InvivoGen (San Diego, CA), and flagellin was purchased from Calbiochem (San Diego, CA). Shiga toxin was supplied by Toxin Technology (Sarasota, FL), and 2,3,7,8-tetrachlorodibenzodioxin (TCDD) was kindly supplied by N. Kaminski (Michigan State University).
Cell cultures.
The mouse macrophage cell line RAW 264.7 (ATCC, Rockville, MD) (2.5 x 105/ml) was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (GIBCO, Chagrin Falls, IL), streptomycin (100 µg/ml), and penicillin (100 U/ml) at 37°C in a humidified atmosphere with 6% CO2. Macrophage cell number and viability were assessed by trypan blue dye exclusion using a hematocytometer. Prior to treatment, cells (2.5 x 105/ml) were cultured in six-well tissue culture plates for 24 h to achieve 80% confluency.
Peritoneal macrophages were elicited by injecting with 1 ml of a sterile 9% (wt/vol) solution of thioglycolate into the peritoneal cavity of 8- to 10-week-old male B6C3F1 (Charles River, Portage, MI) mice (Conrad et al., 1981). After 3 days, mice were euthanized, and the peritoneal cavity was repeatedly flushed with cold Hank's balanced salt solution (HBSS). Pooled peritoneal lavage fluids were centrifuged at 450 x g for 5 min. Cell pellets were washed twice with cold HBSS and then resuspended in supplemented DMEM. Cells were then plated in six-well tissue culture dishes at a cell concentration of 1 x 106/ml. For time course studies, macrophages were incubated for 2 h at 37°C, medium replaced, and adherent cells directly treated with LPS. For LPS concentration studies, macrophages were incubated for 6 h at 37°C, medium replaced, adherent cells incubated for an additional 16 h at 37°C. Medium was replaced and then cultures treated with LPS.
For human whole-blood cultures, heparinized venous blood from healthy donors was diluted (1:10) and cultured in RPMI-1640 medium, 2mM glutamine, 100 U/ml penicillin, and streptomycin (100 µg/ml) as previously described (DeGroote et al., 1992).
Experimental design.
In initial studies focusing on LPS exposure time, LPS concentration, and human cell sensitization (Figs. 1–3), RAW 264.7 cells, peritoneal macrophages, or whole-blood cultures were incubated for a specified time interval with LPS at selected concentrations (0.1–100 ng/ml) or water vehicle (VEH). This was followed by direct addition of DON (250 ng/ml) or water vehicle (VEH) and an additional incubation for 2 h (macrophage cultures) or 6 h (whole-blood cultures) before harvesting for real-time PCR.
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In subsequent studies comparing TLR agonists and evaluating toxicants, RAW 264.7 cultures were (1) incubated for 16 h with LPS or alternative TLR agonist, (2) washed twice, (3) replenished with fresh medium containing DON or other toxicant, (4) incubated for an additional 6 h, and then (5) subjected to RT-PCR. Concentrations of TLR agonists were selected based on previous reports (Alexopoulou et al., 2001
; Jurk et al., 2002; Liaudet et al., 2003; Mutwiri et al., 2003; Ozinsky et al., 2000) as follows: zymosan, 10 µg/ml (TLR2); poly (I:C), 25 µg/ml (TLR3); LPS, 100 ng/ml (TLR4); flagellin, 100 ng/ml (TLR5); R848, 1 µg/ml (TLR7/8); and ODN1826, 1µM (TLR9). Toxicant concentrations were as follows: satratoxin G, 100 ng/ml; Shiga toxin, 1 ng/ml; zearalenone, 100nM; NiCl2, 50µM; TCDD, 10nM; TPT, 1µM; and DNCB, 20µM.
All TLR agonist, toxin, and toxicant concentrations employed were verified to be nontoxic in RAW 264.7 cells using the MTT cleavage test (Marin et al., 1996).
RNA extraction and cytokine mRNA quantitation.
Total RNA from RAW 264.7 cells and peritoneal macrophages was extracted with TRIzol Reagent (Invitrogen, Gaithersburg, MD). Total RNA from whole-blood cultures was isolated using the RNAqueous kit (Ambion Inc, Austin, TX). Resultant RNA was dissolved in nuclease-free water (Promega, Madison, WI) and stored at –80°C. Quantitative PCR reactions for IL-1ß, IL-6, and TNF- mRNA as well as 18S rRNA were performed on an ABI PRISM 7900HT Sequence Detection System using TaqMan One-Step RT-PCR Master Mix and Assays-on-Demand (PE Applied Biosystems, Foster City, NY). Ct values for IL-1ß, IL-6, TNF-, and 18S rRNA were adjusted using the standard curves of known amounts of total RNA (ranging from 0.5 to 500 ng per reaction) and normalized to 18S values (PE Applied Biosystems User Bulletin No. 2).
Statistics.
Data were subjected to one-way ANOVA and post hoc analysis using the SigmaStat for Windows (Jandel Scientific, San Rafael, CA). Data sets showing significant differences were further analyzed for interaction between TLR agonist and toxicant by two-way ANOVA (Slinker, 1998). Groups were considered significantly different when p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LPS Priming Sensitizes Transformed and Primary Macrophages to DON-Induced Proinflammatory Cytokine mRNA Expression
The effects of preincubation with LPS (100 ng/ml) for various time periods on DON-induced proinflammatory gene expression were first assessed in RAW 264.7 cultures. Cells exposed to LPS for 4 or 8 h and then to vehicle for 2 h exhibited moderately increased IL-1ß (Fig. 1A), IL-6 (Fig. 1B), and TNF-
(Fig. 1C) mRNAs compared to cells primed with vehicle. All three cytokine mRNAs were elevated after 16 h but were markedly lower than the previous two time points, suggesting that LPS-induced cytokine responses were transient. DON alone induced very small increases in IL-1ß and TNF- mRNA at all three time points, whereas IL-6 was unaffected. In contrast, cells primed with LPS for 4–16 h and then treated with DON (250 ng/ml) for 2 h exhibited robust IL-1ß (Fig. 1A), IL-6 (Fig. 1B), and TNF- (Fig. 1C) responses that were 1.9- to 5.0-fold, 3.0- to 3.5-fold, and 1.7- to 2.0-fold higher, respectively, than LPS alone. Cell responses were markedly higher than those treated with DON alone. DON-induced cytokine responses in primed cells were significantly higher than the predicted additive responses for all three exposure times (Figs. 1A–1C).
Cytokine responses to DON were enhanced in peritoneal macrophage cultures following LPS priming. The most dramatic effect was on IL-1ß expression, which was elevated in cultures primed with LPS for 4, 8, and 16 h by 40-, 8-, and 6-fold, respectively, compared to LPS alone and 20-, 4-, and 3-fold, respectively, compared to DON alone (Fig. 1D). DON induced a robust IL-6 response in peritoneal macrophages which was elevated 3.4- and 1.6-fold by LPS priming at 4 and 8 h, respectively (Fig. 1E). TNF- mRNA expression was also modestly amplified in peritoneal macrophages primed with LPS and then treated with DON (Fig. 1F). DON-induced responses in LPS-primed cells were significantly higher than predicted additive responses for IL-1ß (4, 8, and 16 h) as well as IL-6 and TNF- (4 h) (Figs. 1D–1F). These data suggest that the capacity of LPS to increase sensitivity to DON was similar in transformed and primary macrophage cultures.
LPS Priming of DON-Induced Proinflammatory Cytokine Expression Is Concentration-Dependent in Transformed and Primary Macrophages
The effects of LPS concentration on the above priming effect was assessed in RAW 264.7 cells by pretreating cultures with 0.1–100 ng/ml LPS for 8 h and then with DON (250 ng/ml) for 2 h. Minimum LPS concentrations for potentiative effects were 1.0, 10, and 1.0 ng/ml for IL-1ß, IL-6, and TNF-, respectively (Figs. 2A–2C). The minimum LPS concentration for induction of all three proinflammatory cytokines in peritoneal macrophages was 1.0 ng/ml (Figs. 2D–2F). Preexposure to 0.1 ng/ml LPS had negligible effects in either cloned or primary culture models (data not shown). DON-induced cytokine responses in primed cells were significantly higher than predicted additive responses at all LPS concentrations used (Figs. 2A–2F). Thus, very low concentrations of LPS could prime both transformed and primary macrophages.
LPS Priming Increases DON-Induced IL-1ß mRNA Expression in Human Blood Cell Cultures
IL-1ß has recently been determined to be selectively upregulated by DON in peripheral blood monocytes (Islam and Pestka, 2006). Since IL-1ß gene expression appeared to be most greatly affected in LPS-primed peritoneal macrophages, the capacity of LPS priming to potentiate a similarly DON-induced IL-1ß mRNA expression was assessed in a human whole-blood culture model. Based on preliminary optimization studies, whole-blood cultures from three individuals were exposed to LPS (5 ng/ml) for 20 h and then to DON (250 ng/ml) for 6 h. Significantly increased IL-1ß mRNAs were observed in LPS-sensitized cultures from all subjects as compared to LPS or DON alone (Fig. 3). There was again a significant interaction between LPS and DON (p < 0.001). Thus, LPS sensitizations of DON-induced IL-1ß response in human monocytes resembled those observed in murine macrophages.
Priming with Other TLR Agonists Sensitizes Macrophages to DON-Induced Proinflammatory Gene Expression
Given the marked effects of LPS priming, the capacity of different TLR agonists to sensitize RAW 264.7 macrophages to DON-induced proinflammatory activation was compared. It was determined in follow-up experiments that priming effects could be further accentuated by using an asynchronous exposure protocol that included (1) a washing step following 16 h of LPS preincubation to remove residual levels of the agonist from the culture and (2) a 6-h DON incubation. This approach was, therefore, employed in this and subsequent comparative studies. Exposures to zymosan (TLR2), poly (I:C) (TLR3), LPS (TLR4), flagellin (TLR5), R848 (TLR7/8), or ODN1826 (TLR9) for 16 h were all capable of sensitizing the cells to DON-induced mRNA expression for IL-1ß (Fig. 4), IL-6 (Fig. 5), and TNF- (Fig. 6). There were significant interactions between individual TLR agonists and DON in all cases except for TLR2 and IL-1ß (Figs. 4–6). These data suggested that activation by many different PAMPs could increase the sensitivity of macrophages to DON.
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LPS Priming Sensitizes Macrophages to Proinflammatory Cytokine Induction by Different Xenobiotics
The capacity of LPS priming to sensitize macrophage to proinflammatory cytokine induction by microbial toxins and anthropogenic toxicants was evaluated. Relative to the microbial toxins, preexposure to LPS for 16 h enhanced IL-1ß, IL-6, and TNF-
expression in RAW 264.7 cells exposed for 6 h to the ribotoxic trichothecene satratoxin (Figs. 7A–7C), the ribotoxic Escherichia coli protein Shiga toxin (Figs. 7D–7F), and the estrogenic mycotoxin zearalenone (Figs.7G–7I). Relative to the anthropogenic toxicants, LPS priming enhanced IL-1ß and IL-6 responses to the metal salts NiCl2 (Figs. 8A and 8B), androgen receptor agonist TPT (Figs. 8D and 8E), Ah receptor agonist TCDD (Figs. 8H and 8I), and the contact sensitizer DNCB (Figs. 8K and 8L) after 6 h, whereas TNF- expression was enhanced only with DNCB (Figs. 8C, 8G, and 8J). Thus, the macrophage sensitization paradigm could be expanded to include an array of toxicants with diverse chemical structures and mechanisms of action.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Innate immune system activation might potentially predispose a host to toxicant-induced tissue injury. LPS amplifies the toxicity of a diverse array of xenobiotic chemicals with cytokines possibly being important mediators of some of these effects (Ganey and Roth, 2001
). The results presented herein suggest that LPS priming renders macrophages sensitive to induction of proinflammatory cytokines by DON and other xenobiotics (Fig. 9). In an in vivo correlate of the present study, we found previously that a single LPS exposure sensitizes the innate immune system of the mouse to DON-induced proinflammatory cytokine expression for at least 24 h as reflected by reduced minimum DON dose required for effect as well as increased magnitude and duration of responses (Islam and Pestka, 2006). These observations and current in vitro findings are of critical importance because they suggest that episodic exposure to LPS and potentially other TLR agonists might induce a state of increased sensitivity to a toxic chemical in a host, ultimately resulting in heightened innate immune system activation and related downstream sequelae.
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One consideration of the present study is the potential for commercial TLR agonists to contain trace levels of LPS (Tsan and Gao, 2004
). Thus, future research that focuses on a specific TLR receptor should verify absence of such contamination in commercial ligand preparations.
While the capacity of TNF- and IL-6 to mediate injurious inflammatory processes is well documented (Schletter et al., 1995), the dramatic elevation of IL-1 gene expression observed might be of particular importance because this cytokine has been previously linked to leukocyte apoptosis (Islam and Pestka, 2003). Notably, exposure of mice to subtoxic doses of LPS and DON causes sequential IL-1ß overexpression, plasma glucocorticoid elevation, and severe apoptotic depletion of the lymphoid tissue (Islam et al., 2002, 2003). LPS and DON elevate and prolong corticosterone levels that drive apoptosis, and this can be attenuated with the glucocorticoid receptor antagonist RU 486 (Islam et al., 2002). Studies using IL-1 receptor knockout mice and IL-1 receptor antagonist have shown that IL-1 is an important mediator of LPS plus DON-induced corticosterone and subsequent leukocyte apoptosis (Islam and Pestka, 2003). Thus, the capacity of TLR agonist–primed macrophages to respond to subsequent xenobiotic challenges with extraordinarily heightened IL-1ß expression might contribute to enhanced leukocyte apoptosis in an exposed host.
The xenobiotics evaluated in this study have diverse mechanisms of action and included microbial toxins and anthropogenic toxicants. The molecular target of trichothecene mycotoxins is the 60S ribosomal subunit, the binding of which causes translational inhibition as well as rapid activation of mitogen-activated protein kinases (MAPKs) in a process termed the "ribotoxic stress response"(Iordanov et al., 1997; Shifrin and Anderson, 1999; Yang et al., 2000). Like DON, the trichothecene satratoxin (Yang et al., 2000) and Shiga toxin (Cherla et al., 2006) drive MAPK activation in the macrophage via interaction with the ribosome. The mycotoxin zearalenone can induce IL-1ß transcription in macrophages via interaction with the estrogen receptor (Ruh et al., 1998). Its capacity to drive cytokine expression following TLR priming suggests that a microbial toxin that does not act by the ribotoxic stress response can stimulate sensitized macrophages.
Based on the findings with the microbial toxins, a variety of anthropogenic toxicants known to induce proinflammatory cytokines were also assessed in TLR-sensitized macrophage, each of which has a different mechanism of action. Nickel and other metals appear to evoke cytokine production in macrophages through the formation of activated oxygen species (Niki et al., 2003). Triphenyltin has been suggested to be an endocrine disruptor that acts through the androgen receptor (Ozinsky et al., 2000). TCDD is a potent agonist of the Ah receptor which has been found with Ah receptor nuclear translocator in transformed and primary macrophages (Komura et al., 2001). DNCB, a contact sensitizer, likely evokes cytokine expression via an MAPK-dependent mechanism involving oxidative stress (Arkusz et al., 2006; Mizuashi et al., 2005). Remarkably, it appears that the priming effects of LPS were dissociable from the type of secondary xenobiotic stressor employed.
Although the underlying mechanisms by which TLR agonists sensitize macrophages to xenobiotics are not known, insights might be drawn on the capacity of diverse chemicals to similarly drive the secondary response. We propose that TLR activation serves as an initial danger signal (Kirk and Bazan, 2005; Matzinger, 2002), thereby reprogramming the macrophage to a phenotype that is exquisitely sensitive to secondary danger signals generated by subsequent exposure to toxicants (Fig. 9). In support of this contention, there is growing evidence for functional plasticity in macrophages as well as for their capacity to adapt reversibly to changing microenvironments (Mantovani et al., 2002; Stout and Suttles, 2004). Macrophages can be selectively reprogrammed to a specific phenotype of immune response following relatively short-term exposure to microbial ligands (Malyshev and Shnyra, 2003). Polarization of human macrophages into proinflammatory type 1 and anti-inflammatory type 2 occurs in response to microbial antigens, and IFN-
– and CD40L-mediated costimulation has been recently reported (Verreck et al., 2006). Depending on the agonist employed, macrophages display different patterns of function relative to cytokines and enzymes produced, and these might not display a strict dichotomy between type 1 and type 2 responses (Stout et al., 2005). Particularly critical to such macrophage reprogramming are the effects of prior exposure to specific cytokines.
It is tempting to speculate that the sensitized macrophage phenotype observed herein is the product of the cytokine milieu generated by treatment with LPS or other TLR agonist. Mechanisms for macrophage reprogramming are as yet poorly understood. In several experiments, we attempted to demonstrate that LPS priming caused prolonged and enhanced MAPK or NF-
B activation without success (data not shown). One alternative attractive mechanism is chromatin remodeling which has been proposed to mediate innate phenotype changes in several immune and inflammatory disease models (Escoubet-Lozach et al., 2002; Lee et al., 2003; Miao et al., 2004; Rahman, 2003; Sullivan, 2003; Zhang et al., 2004). During chromatin remodeling, histone acetylation and phosphorylation render cytokine gene promoters accessible to transactivation. Enhanced promoter access in TLR agonist–primed macrophages could facilitate rapid, robust cytokine responses upon exposure to secondary stress signals by xenobiotics chemicals, even those having different mechanisms of action as were observed here.
Taken together, the in vitro results presented herein suggest that TLR activation might sensitize the innate immune system to xenobiotic chemicals. Further studies of TLR priming should focus on the roles of phenotypic differentiation in the sensitization of macrophage to chemical stressors as well as the potential involvement of chromatin remodeling. Interestingly, TLRs have now been identified as therapeutic targets for a number of diseases (McInturff et al., 2005). Synthetic TLR agonists such as imiquimod, which activates TLR 7/8, are currently being used clinically to treat skin diseases. Such a human population might have increased sensitivity to chemical toxicants. It might thus be important to assess the peripheral blood mononuclear population of such patients for increased responsiveness to xenobiotic chemicals.
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NOTES |
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Disclaimer: The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service Grants ES03553 and DK058833 from the National Institutes for Health. We would like to thank K. Penner, J. Gray, and A. Thelen for technical assistance and M. Rosner and R. Roth for help with manuscript preparation.
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