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12,14-Prostaglandin J2
Mie Mochizuki,3, Yukio Ishii,3 Tetsuro Ishii,4 Takahiro Shibata,5 Yoshiyuki Kawamoto,5 Vincent Kelly,1,2 Kiyohisa Sekizawa,3 Koji Uchida,4 and Masayuki Yamamoto1,2*
ERATO Environmental Response Project,1 Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance,2 Institute of Clinical Medicines,3 Institute of Social Medicines, University of Tsukuba, Tsukuba 305-8577,4 Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan5
Received 25 April 2003/ Returned for modification 17 July 2003/ Accepted 26 September 2003
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ABSTRACT |
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12,14-prostaglandin J2 (15d-PGJ2), it was observed that macrophages from pleural lavage accumulate 15d-PGJ2. We show that in mouse peritoneal macrophages 15d-PGJ2 can activate Nrf2 by forming adducts with Keap1, resulting in an Nrf2-dependent induction of heme oxygenase 1 and peroxiredoxin I (PrxI) gene expression. Administration of the cyclooxygenase 2 inhibitor NS-398 to mice with carrageenan-induced pleurisy caused persistence of neutrophil recruitment and, in macrophages, attenuated the 15d-PGJ2 accumulation and PrxI expression. Administration of 15d-PGJ2 into the pleural space of NS-398-treated wild-type mice largely counteracted both the decrease in PrxI and persistence of neutrophil recruitment. In contrast, these changes did not occur in the Nrf2-deficient mice. These results demonstrate that Nrf2 regulates the inflammation process downstream of 15d-PGJ2 by orchestrating the recruitment of inflammatory cells and regulating the gene expression within those cells. |
INTRODUCTION |
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Nrf2 is a member of the leucine zipper transcription factor family, and its activity is pivotal for the coordinate induction of phase II detoxifying and antioxidative enzymes whose expression is under the regulatory influence of ARE (15, 16, 18, 41). Nrf2 thus contributes to cytoprotection against environmental electrophiles and oxidative stresses (1, 4, 7, 11, 16, 33). Consistent with its assigned role in protection from environmental stress, Nrf2 is highly expressed in detoxication organs, including the gastrointestinal tract, liver, kidney, and lung. In addition, Nrf2 is abundantly expressed in activated macrophages, thyroid glands, and brown adipose tissue, suggesting additional physiological roles beyond detoxication (5).
The inflammatory response requires a coordinated integration of various signaling pathways, including cyclooxygenases (COX), nitric oxide, and cytokines (28, 40). COX enzymes catalyze the conversion of arachidonic acid to prostaglandin H2 (PGH2), from which other prostaglandins (PGs) are derived by the concomitant action of a variety of PG synthetases. Of the COX enzymes, COX-2 is found mainly in inflammatory cells and tissues. COX-2 is found to be upregulated during acute inflammation (37). By producing PGH2, COX-2 promotes the synthesis of PGE2, an important component of the inflammatory cascade that manifests many of the cardinal signs of inflammation (10). Intriguingly, since COX-2 is also expressed in the late phase of inflammation, it is widely accepted that COX-2 is associated with the resolution, as well as the establishment, of the acute inflammatory response (13). However, in contrast to the case during early stages of inflammation, pleural exudates of rats taken during the resolution stage of inflammation were found to contain high concentrations of PGD2 and 15-deoxy-12,14-PGJ2 (15d-PGJ2) with minimal levels of PGE2 expression.
PGs can be divided into two subtypes: conventional PGs and cyclopentenone PGs (cyPGs) (44). Conventional PGs, such as PGE2 and PGD2, bind to cell surface receptors to exert their actions. However, no cell surface receptors have been identified for cyPGs, such as 15d-PGJ2 and PGA2. Rather, cyPGs are actively transported into cells, where they accumulate in nuclei and act as potent repressors of cell growth and inducers of cell differentiation (12, 29). It has been reported that 15d-PGJ2 exerts its anti-inflammatory activity through activation of peroxisome proliferator-activated receptor 
(PPAR) (21, 34) or by directly inhibiting nuclear factor kappa B (NF-
B) activation by binding covalently to the IB kinase (36).
Recently, Nrf2 target genes in macrophages were suspected of playing anti-inflammatory roles. For instance, HO-1 exerts anti-inflammatory functions in various systems, including carrageenan-induced pleurisy, through generation of carbon monoxide (30, 43). Furthermore, human peroxiredoxin I (PrxI or PAG) was recently identified as a negative regulator of macrophage migration inhibitory factor (MIF) (22), a crucial factor in the regulation of inflammation (26) and sepsis (35). These observations led us to explore possible roles for Nrf2 in the acute inflammatory response. To this end, we exploited carrageenan-induced pleurisy in mice as a model system. The results of this study reveal that the Nrf2-ARE system regulates the acute inflammation process by orchestrating the recruitment of inflammatory cells. We also found that COX-2 mediates the intracellular accumulation of 15d-PGJ2 and that 15d-PGJ2, which is shown to activate Nrf2, in turn regulates the expression of PrxI and other anti-oxidative stress enzymes in activated inflammatory macrophages.
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MATERIALS AND METHODS |
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RT-PCR analysis. Total RNAs (1 µg) were reverse transcribed into cDNA and used for a reverse transcription-PCR (RT-PCR) analysis (Qiagen, Hilden, Germany). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a positive control. The PCR products were separated in a 1.5% agarose gel, and positive signals were quantified by densitometry analysis after staining with ethidium bromide.
Immunoblotting. The nuclei of peritoneal macrophages were solubilized with sodium dodecyl sulfate (SDS) sample buffer without loading dye and 2-mercaptoethanol, and protein concentrations were estimated by the bicinchoninic acid protein assay (Pierce, Rockford, Ill.). Proteins were separated by SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol and electrotransferred onto Immobilon membranes (Millipore). To detect immunoreactive proteins, we used horseradish peroxidase-conjugated anti-rabbit immunoglobulin G and ECL blotting reagents (Amersham). An anti-Nrf2 antibody was used as previously described (16). Inflammatory cell pellets from pulmonary lavage were lysed by sonication in buffer containing 50 mM Tris-HCl (pH 7.4), 25 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1% Nonidet P-40, and protease inhibitors. After centrifugation at 8,000 x g for 5 min at 4°C, protein concentrations in the supernatants were determined by the Bio-Rad protein assay. Samples were boiled with gel loading buffer (62.5 mM Tris-HCl, 2% SDS, 25% glycerol, and 0.01% bromophenol blue) at a ratio of 1:1 for 5 min. Total protein equivalents for each sample were separated by SDS-5 to 15% polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol and were transferred to Sequi-Blot polyvinylidene difluoride membranes (Bio-Rad). Blots were incubated with polyclonal rabbit antibody against murine PrxI (17).
Carrageenan-induced pleurisy. Wild-type and nrf2-/- mice of the ICR/129SV background weighing 20 to 25 g were used throughout the experiments. A 0.25% lambda carrageenan solution in saline (0.1 ml) was injected into the right pleural cavities of the animals. At 2, 6, 12, 24, 48, and 72 h and 7 days after the injection of carrageenan, the chest was carefully opened and the pleural cavity was washed with 1 ml of saline solution containing heparin. The pleural cavity was washed five times consecutively in the same way, and the pleural lavage fluid was collected into a tube.
Leukocyte counts. A 30-µl sample of collected pleural lavage fluid was diluted with Turk's solution, and total leukocytes were counted under an optical microscope. Differential leukocyte counts were determined in cytospin smears stained with Wright-Giemsa stain (Diff-Quick; Sysmex, Kobe, Japan).
Albumin concentration. The first washing was centrifuged at 400 x g for 5 min at 4°C, and the albumin concentration in the supernatant was determined with the albumin reagent from Sigma (St. Louis, Mo.).
Immunohistochemical analysis. Cells were smeared onto poly-L-lysine-coated slides and allowed to air dry. Endogenous peroxidases were quenched with 0.3% H2O2 in methanol, and sections were washed with 0.1% Triton X-100 in phosphate-buffered saline. The sections were reacted with anti-15d-PGJ2 monoclonal antibody (38), anti-PrxI antibody (16), anti-HO-1 antibody (a generous gift from Shigeru Taketani), anti-F4/80 antibody (Serotec), anti-COX-2 antibody (Santa Cruz), or anti-hematopoietic PG synthetase (PGDS) antibody (Santa Cruz) and incubated for another hour with Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan). Diaminobenzidine was used as a chromogen.
NS-398 and indomethacin treatment. NS-398 (Cayman Chemical, Ann Arbor, Mich.) (10 mg/kg) and indomethacin (Sigma) (10 mg/kg) were administered intraperitoneally 1 h before the injection of carrageenan. The pleural cavity was washed at 2, 12, and 24 h after the injection of carrageenan for the determination of inflammatory cell numbers and albumin concentration. To determine the effects of NS-398 at 48 and 72 h and 7 day, NS-398 was administered every 24 h thereafter. At 48 h, 72 h, and 7 days after the injection of carrageenan, the pleural cavity was washed for the determination of inflammatory cell numbers and albumin concentration.
15d-PGJ2 administration. At 1 h after the intraperitoneal injection of NS-398, 15d-PGJ2 (100 µg/kg) was injected into the pleural cavity. At 24 h after the injection of carrageenan, the pleural cavity was washed for the determination of inflammatory cell numbers and anti-inflammatory gene expression levels.
Statistical analysis. Statistical analysis was done by analysis of variance followed by a Bonferroni posttest. Albumin concentration data were analyzed by using Welch's t test. A P value of less than 0.05 was accepted as statistically significant.
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RESULTS |
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15d-PGJ2 accumulates in pleural inflammatory macrophages. Since 15d-PGJ2 has been implicated as a key regulator of carrageenan pleurisy (8, 13, 44), we examined whether 15d-PGJ2 accumulates in pleural inflammatory cells by using a specific monoclonal antibody against 15d-PGJ2. The antibody has successfully detected the accumulation of 15d-PGJ2 in both RAW264.7 cells activated by lipopolysaccharide and foamy macrophages of human atherosclerotic lesions (38). Furthermore, this antibody has shown to react almost exclusively with 15d-PGJ2 (38). Immunohistochemical analysis with this antibody revealed that accumulation of 15d-PGJ2 specifically occurred in pleural macrophages that were positive for F4/80 antigens but did not occur in neutrophils (data not shown).
As reported previously for rat carrageenan pleurisy, the accumulation of 15d-PGJ2 showed two peaks (13). The accumulation of 15d-PGJ2 was transiently observed at 2 h of pleurisy (Fig. 2A), but the accumulation then decreased at 6 h of pleurisy (Fig. 2B). At 2 h of pleurisy, the inducible accumulation of 15d-PGJ2 in macrophages appeared to occur strongly around the nuclear membranes of resident pleural macrophages, which are small and round. The level increased again at 12 h after the carrageenan injection and remained high until 48 h of pleurisy (Fig. 2C and data not shown), followed by another increase at 72 h of pleurisy (Fig. 2D). At the 24-h time point, the accumulation of 15d-PGJ2 was observed mainly in the cytoplasm of macrophages, which were large and had a foamy appearance (Fig. 2C). These results thus demonstrate that 15d-PGJ2 specifically accumulates in pleural inflammatory macrophages and suggest that 15d-PGJ2 may act through modifying macrophage function.
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,ß-unsaturated carbonyl group in the cyclopentane ring. This ring structure renders this molecule capable of forming Michael adducts with nucleophilic cellular molecules and covalent modification of specific proteins. We speculate that this feature of cyPGs may activate Nrf2 (39). In order to obtain solid evidence for activation of the Nrf2 pathway by 15d-PGJ2, we first examined the effect of exogenous 15d-PGJ2 on the induction of three Nrf2 target genes in primary cultures of mouse peritoneal macrophages, which were used previously for the study of Nrf2 (16). The results clearly indicated that 15d-PGJ2 activates, in a dose-dependent manner, the expression of the HO-1, PrxI, and A170 genes (Fig. 3A), all of which were shown to be inducible by electrophiles in peritoneal macrophages (16).
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, 15d-PGJ2, thromboxane B2, and leukotriene B4, only PGA1, 15d-PGJ2, and PGD2 markedly induced the expression of Nrf2 target genes (Fig. 3B). PGA1 and 15d-PGJ2 are classified as electrophilic cyPGs. Since PGD2 is readily metabolized to 15d-PGJ2 (38), 15d-PGJ2 is suggested to be the most important cyPG in the regulation of Nrf2. cyPGs activate Nrf2 in macrophages and hepatocytes. Subsequently, we examined the requirement for Nrf2 in the induction of antioxidant genes, using primary cultures of peritoneal macrophages from Nrf2-deficient mice. While HO-1, PrxI, and A170 mRNAs were all induced in wild-type macrophages by the addition of 5-µM 15d-PGJ2 to the culture medium (Fig. 4A, lane 2), the induction was not observed in nrf2-/- macrophages (lane 6). The result was reproducible with 10-µM 15d-PGJ2 (lanes 3 and 7), but in this case a weak induction was observed in the nrf2-/- macrophages (lane 7), suggesting the presence of a minor complementary pathway to Nrf2. Consistent with these results, immunoblot analysis with anti-Nrf2 antibody revealed that 15d-PGJ2 (Fig. 4B, lane 3) and PGA1 (lane 4), but not PGE2 (lane 5), induced the nuclear accumulation of Nrf2.
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Recent studies indicate that certain Nrf2 inducers, such as Michael reaction acceptors, directly bind to reactive cysteine residues in Keap1, thereby liberating Nrf2 (9). To elucidate whether 15d-PGJ2 binds directly to Keap1, we examined the binding of biotin-tagged 15d-PGJ2 to Keap1 in RL34 cells. A pull-down analysis with avidin beads followed by probing with anti-Keap1 antibody demonstrated that Keap1 could be detected in precipitates from biotinylated 15d-PGJ2-treated or biotinylated 12-PGJ2-treated cells but not in the control immunoprecipitates (Fig. 4E). This result suggests that 15d-PGJ2 directly binds to Keap1, most probably through covalent linkage, allowing Nrf2 to be released from Keap1.
COX-2 inhibitor affects the inflammatory cell infiltration. In order to elucidate the relationship between the accumulation of 15d-PGJ2 and activation of Nrf2, we examined the time course of PrxI gene expression during the carrageenan-induced pleurisy. Immunoblot analyses with anti-PrxI antibody revealed that the expression of PrxI was induced at as early as 2 h of pleurisy (Fig. 5A, lanes 3 to 5). In contrast, the induction of PrxI during pleurisy was largely abolished in nrf2-/- mice, and this difference was prominent at 2 to 24 h (lanes 4, 7, and 10) but became relatively small after 48 h of pleurisy (lanes 13 and 16).
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Immunohistochemical analysis demonstrated that PrxI expression was specifically observed in macrophages, but not in neutrophils, of pleural lavage fluid (Fig. 5B). The expression profile of PrxI clearly overlapped with those of 15d-PGJ2 and macrophage-specific F4/80 antigen (Fig. 5B). The expression of HO-1 was also detected exclusively in pleural macrophages (Fig. 5B), in very good agreement with previous analysis of the rat pleurisy model. Furthermore, COX-2 and hematopoietic PGDS, two major rate-limiting enzymes for 15d-PGJ2 synthesis, were highly expressed in the macrophages (Fig. 5B), supporting our contention that 15d-PGJ2 was synthesized mainly in the macrophages.
It should be noted that NS-398 treatment caused a persistence of neutrophil infiltration at 24 h of pleurisy (Fig. 5C) and a delay in macrophage recruitment (Fig. 5D) in pleural lavage fluid. This is in contrast to the case for untreated mice, where the macrophage number was high at 48 h and decreased at 72 h of pleurisy. It can be observed that in the NS-398-treated mice the macrophage number continued to climb even at 72 h of pleurisy (Fig. 5D). Also, in the NS-398-treated mice, the macrophage number was rather low at the 24- and 48-h time points, indicating a delay in macrophage recruitment. The persistence of neutrophil infiltration and the delay of macrophage recruitment were also observed in the COX-1/COX-2 dual inhibitor indomethacin (Fig. 5E). Thus, inflammatory cell infiltration in COX-2-inhibited mice closely reflects that observed in Nrf2-deficient mice, further supporting our contention that 15d-PGJ2 (and COX-2) acts as a regulator of the Nrf2 pathway and the expression of antioxidant genes.
Replacement of 15d-PGJ2 into the intrapleural space. Finally, we wished to test directly the significance of 15d-PGJ2 accumulation in the development of and recovery from carrageenan-induced pleurisy. To this end, we administered 15d-PGJ2 into the intrapleural cavity and examined changes in inflammatory cell infiltration at 24 h of pleurisy. Consistent with the results shown above, administration of NS-398 to mice with pleurisy increased the neutrophil number at 24 h (Fig. 6A, bar 2). We found that simultaneous injection of 15d-PGJ2 with carrageenan into the pleural space reversed the increase of neutrophil infiltration in NS-398-treated mice (bar 3). The important finding here is that the administration of 15d-PGJ2 into the pleural space of Nrf2-null mice did not affect the accumulation of neutrophils (compare bars 4 and 5).
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To determine changes in the antioxidant gene expression, we carried out RT-PCR analyses with pleural inflammatory cells and specific primers for PrxI (Fig. 6C) and HO-1 (Fig. 6D). The expression of PrxI and HO-1 showed a pattern of changes similar to that for macrophage number. The NS-398-treatment produced a decrease in PrxI and HO-1 gene expression (bar 2). On the other hand, the administration of 15d-PGJ2 to the NS-398-treated mice reversed the PrxI and HO-1 mRNA expression level (bar 3) to that for wild-type control mice (bar 1). The expression levels of PrxI and HO-1 mRNAs in the Nrf2-deficient mice did not change with 15d-PGJ2 treatment (compare bars 4 and 5). These results thus argue that 15d-PGJ2 transduces the inflammatory signals to Nrf2 and that Nrf2 transcriptionally regulates both the antioxidant gene expression in pleural macrophages and inflammatory cell recruitment.
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DISCUSSION |
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Increasing lines of evidence (8, 44, 45) suggest that in addition to establishing acute inflammation, COX-2 also contributes to the resolution phase of inflammation through 15d-PGJ2. 15d-PGJ2 can function as both an activator and a repressor of signal-transducing transcription factors. The possible contributions made by 15d-PGJ2 to the acute inflammatory response are summarized in Fig. 7. It has been reported that 15d-PGJ2 inhibits NF-B activation by covalently binding to IB kinase ß or the p50 subunit of NF-B (3, 36). In addition, 15d-PGJ2 has been reported to serve as a natural ligand of PPAR (21, 34). Activated PPAR regulates the synthesis of proinflammatory cytokines and the induction of nitric oxide synthetase in activated monocytes by negatively interacting with AP-1, NF-B, or STAT. Thus, 15d-PGJ2 may have an impact upon multiple mechanisms during the resolution of inflammation. The results of this study demonstrate that 15d-PGJ2 can act as a potent anti-inflammatory agent by exploiting the Nrf2-Keap1 pathway, a previously unrecognized alternative pathway in the cascades downstream of 15d-PGJ2.
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)-mediated induction of vascular cell adhesion molecule-1 gene expression, which is important for monocyte recruitment during inflammation response. Our present observations, along with those cited above, suggest that Nrf2 is important for regulating the process of acute inflammation.
We demonstrated in this study that the intracellular accumulation of 15d-PGJ2 occurs during carrageenan-induced pleurisy in the mouse (Fig. 2). This observation is consistent with the previous analysis in rat carrageenan pleurisy, where 15d-PGJ2 accumulates with two peaks at low nanomolar levels in the pleural exudates. As the 15d-PGJ2 concentration used in this study is higher than that detected in pleural exudates, it might be challenging to hypothesize that 15d-PGJ2 works as an endogenous regulator of acute inflammation. However, Narumiya et al. previously reported that exogenously added 12-PGJ2 accumulates in the cells in a protein-bound form and is resistant to cell extraction, suggesting that intracellular sequestration of these cyPGs is most probably due to the Michael adduction to the protein (27). Furthermore, Keap1 localizes in the perinuclear cytoskeleton close to COX-2 and PGDS. Therefore, it is likely that the local concentration of cyPGs accumulates to a level sufficient for Nrf2 activation during pleurisy.
It was reported recently that submicromolar concentrations of 15d-PGJ2 can activate HO-1 and contribute to the anti-inflammatory effect of this reagent (25). As NF-B inhibition requires a concentration of 15d-PGJ2 in the micromolar range in cell culture (36), this observation suggests that an in vivo anti-inflammatory effect of 15d-PGJ2 may be more relevant to the activation of Nrf2.
Under normal conditions, Nrf2 activity is suppressed primarily by its compartmentalization to the cytosol by Keap1 and consequent rapid degradation by the proteasome (reference 20 and references therein). Recently it was demonstrated that electrophiles classified as Michael reaction acceptors directly bound to Keap1 and dissociated Nrf2 from Keap1 (9). We have demonstrated in this study that 15d-PGJ2 and 12-PGJ2, both of which contain a reactive ,ß-unsaturated carbonyl group in their cyclopentane ring, directly bind to Keap1 and activate Nrf2. These results are consistent with our contention that cyPGs act as endogenous activators of the Nrf2-Keap1 pathway in macrophages, thereby regulating the recruitment of inflammatory cells.
NS-398-treated wild-type mice and nrf2-null mice displayed similar phenotypes in pleurisy, i.e., a persistence of neutrophil residence and a delay in macrophage recruitment. These results suggest that COX-2 products negatively regulate the accumulation of neutrophils in the intrapleural space, through either enhancing the phagocytosis of neutrophils by macrophages or decreasing the neutrophil infiltration. The administration of 15d-PGJ2 into the pleural space successfully reversed the phenotype in NS-398-treated mice, indicating that 15d-PGJ2 works downstream of COX-2 activation. Consistent with this observation, we demonstrated in this study that the actual inducible accumulation of 15d-PGJ2 occurred in macrophages during carrageenan pleurisy (Fig. 2). Since the introduction of 15d-PGJ2 into the pleural spaces of Nrf2-deficient mice had no effect, Nrf2 must be a downstream mediator of 15d-PGJ2 activity in macrophages. Thus, we conclude that 15d-PGJ2 regulates acute inflammation through regulating the function of macrophages (Fig. 7).
It remains to be elucidated to how Nrf2 regulates acute inflammation. We speculate that a battery of Nrf2 target genes cooperatively function to repress proinflammatory signals, such as those of TNF- or interleukin-1ß. HO-1 and PrxI are the Nrf2 target genes that are likely to influence the inflammatory process (Fig. 7) (16). Recently, Gong et al. have demonstrated that 15d-PGJ2 can activate HO-1 via a stress-responsive element and by an Nrf2-mediated mechanism (14). In the carrageenan-induced pleurisy model, it has been shown that elevation of HO-1 expression can suppress, whereas inhibition of HO-1 activity can exacerbate, the inflammatory response (43, 45). Indeed, HO-1 can inhibit the expression of TNF- most probably through the generation of carbon monoxide (30). With respect to PrxI, its human counterpart PAG was reported to directly bind to and negatively regulate ß-tautomerase activity of MIF, which is one of the central regulators of inflammation (22). Although the physiological significance of the ß-tautomerase activity of MIF is unclear at present, we nonetheless expect that the repression of MIF activity by PrxI may play important roles in the regulation of inflammation. Furthermore, the overexpression of PrxI removed H2O2 (23), suggesting that PrxI can repress TNF- signaling by the removal of H2O2 (Fig. 7). These observations suggest that Nrf2 may have multiple downstream targets that regulate the acute inflammation process.
In the rat carrageenan-induced pleurisy model, accumulation of 15d-PGJ2 in the late phase of pleurisy was associated with resolution of the inflammation (13). However, the true role of 15d-PGJ2 during the early phase of pleurisy remained largely unknown. Our results imply that in the early phase, accumulation of 15d-PGJ2 activates Nrf2 and regulates the inflammation process through the induction of target gene expression, including that of HO-1 and PrxI. Whereas COX-2 has been reported to accelerate inflammation in the early phase of pleurisy through the induction of PGE2, our present analyses suggest that COX-2 also can suppress the early phase of inflammation through the production of 15d-PGJ2. These results are consistent with the present model that the inflammation process is balanced by an acceleration and deceleration of integrating signaling pathways (28). Understanding of how the signals are integrated to establish and resolve the acute inflammation process provides important clues to facilitate the development of effective treatments for chronic inflammation.
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ACKNOWLEDGMENTS |
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This work was supported in part by grants from JST-ERATO; the Ministry of Education, Culture, Sports, Science and Technology; the Ministry of Health, Labor and Welfare; and the Naito foundation.
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FOOTNOTES |
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K.I. and M.M. contributed equally to this work.
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