doi:10.1182/blood-2012-04-426247
Prepublished online July 12, 2012;
2012 120: 2428-2437
Mark Boldin and David Baltimore
Alex Yick-Lun So, Yvette Garcia-Flores, Aarathi Minisandram, Ayana Martin, Konstantin Taganov,
by Bach1/HO-1 pathway in mice
Regulation of APC development, immune response, and autoimmunity
http://bloodjournal.hematologylibrary.org/content/120/12/2428.full.html
Updated information and services can be found at:
(4866 articles)
Immunobiology
Articles on similar topics can be found in the following Blood collections
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests
Information about reproducing this article in parts or in its entirety may be found online at:
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints
Information about ordering reprints may be found online at:
http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml
Information about subscriptions and ASH membership may be found online at:
Copyright 2011 by The American Society of Hematology; all rights reserved.
Washington DC 20036.
by the American Society of Hematology, 2021 L St, NW, Suite 900,
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly
IMMUNOBIOLOGY
Regulation of APC development, immune response, and autoimmunity by
Bach1/HO-1 pathway in mice
Alex Yick-Lun So,1Yvette Garcia-Flores,1Aarathi Minisandram,1Ayana Martin,1Konstantin Taganov,1Mark Boldin,2and
David Baltimore1
1Department of Biology, California Institute of Technology, Pasadena, CA; and2Department of Molecular and Cellular Biology, Beckman Research Institute of the
City of Hope, Duarte, CA
APCs are essential for innate and adap-tive immunity as well as self-immune tolerance. Here, we show that the Cap’n’collar member Bach1 regulates the generation of APCs, specifically macro-phages and dendritic cells, in mice. The impaired APC development in Bach1ⴚ/ⴚ mice was accompanied by defects in downstream T-cell responses and partial
protection from experimental autoim-mune encephalomyelitis. Genomewide analyses identified a panel of Bach1 tar-get genes and ablation of the direct Bach1 target gene HO-1 exacerbated the im-paired APC development observed in Bach1ⴚ/ⴚmice. This was attributed to the impaired ability of HO-1ⴚ/ⴚBach1ⴚ/ⴚ double mutants to produce upstream APC
progenitor cells, including common my-eloid progenitor (CMP)–Flk2ⴙ. By con-trast, we observed an increase in hemato-poietic stem-progenitor cells (HSPCs) in these mice, suggesting a developmental block in the progression of HSPCs to CMP-Flk2ⴙ and subsequently APCs. (Blood. 2012;120(12):2428-2437)
Introduction
Abnormal development of APCs can result in immunodeficiency and autoimmune disease. APCs, which include macrophages, B cells, and dendritic cells (DCs), and are critical for mediating adaptive immunity to foreign antigens as well as inducing tolerance to self-antigens. At steady state, macrophages are generated from monocytes, and classic DCs are derived from their precursor (pre-cDCs) and common DC progenitors (CDPs).1-6 These cells
are all generated from upstream macrophage-DC precursor (MDP) cells,7-9which are themselves derived from less lineage-restricted
common myeloid progenitors (CMPs).1The entire populations of
hematopoietic cells originate from pluripotent HSCs in the BM (see Figure 7A). Despite recent advances, the cellular factors that regulate the progression of HSCs to the development of APCs remain largely uncharacterized.
Cap ‘n’ collar (CNC) proteins are evolutionarily conserved factors that are known to be essential for the resolution of oxidative stress.10 In Caenorhabditis elegans, the CNC protein SKN-1
regulates longevity.11,12In mammals, there are 6 members within
the CNC family (p45, Nrf1, Nrf2, Nrf3, Bach1, and Bach2) that heterodimerize with small Maf proteins (MafK, MafG, and MafF) to direct either gene induction or repression.13 For instance,
although the co-occupancy of p45 and MafK at genome elements activates transcription, binding of a Bach1 and MafK heterodimer to these elements results in gene repression.14Many different CNC
and Maf heterodimers are formed,15causing the biology of CNC
and Maf members to be complex. Although Nrf1⫺/⫺ mice are anemic and have embryonic or postnatal lethality,16ablation of p45
in mice leads to defective platelet production.17Although deletion
of Bach2 in mice caused impaired antibody class switching,18
disruption of Nrf2 resulted in a neurodegenerative disorder and autoimmune disease.19-21Deciphering the physiologic role of some
of the CNC/Maf proteins has also proven to be challenging because of the potential functional redundancy among them.22,23
The CNC/Maf network has been associated with a myriad of human disorders, including those of the skin, respiratory system, and hematopoietic system.10,15,24Thus, drug therapy involving the
CNC pathway is actively being studied in humans for cancer chemoprevention, inflammatory diseases, and autoimmune dis-eases (for review, see Sykiotis and Bohmann10). For example, the
fumaric acid ester dimethylfumarate, which exerts neuroprotection by inducing Nrf2 expression, is currently in a phase 3 clinical trial as an orally active effective treatment of human multiple sclerosis with limited side effects.25,26Thus, deciphering the functions of the
individual CNC subunits will not only be crucial to unraveling the overlapping and unique functions of these proteins but also provide insights to the potential of targeting CNC proteins for therapy against relevant diseases. In particular, the CNC member Bach1 is up-regulated in fetal Down syndrome (DS) brain.27Bach1
expres-sion is also significantly higher in patients with DS–acute mega-karyoblastic leukemia than patients without DS– acute megakaryo-blastic leukemia.24 The best-known effect of Bach1 on
transcriptional regulation is its repression of heme oxygenase-1 (HO-1),14,28,29which is a pivotal enzyme for metabolism of heme
for iron reutilization and resolving oxidative stress.30,31 HO-1
inhibits the development of autoimmune neuroinflammation prob-ably by suppressing APC function and reducing the accumulation of self-reactive T cells in the CNS.32At steady state, Bach1 directly
binds to the HO-1 promoter to repress expression. During condi-tions of high heme concentration, heme associates with Bach1 and relieves this repressive effect and results in HO-1 up-regulation. In theory, the Bach1/HO-1 pathway serves as a feedback loop to maintain homeostasis of heme and to prevent oxidative stress.33
Submitted April 25, 2012; accepted July 7, 2012. Prepublished online as Blood First Edition paper, July 12, 2012; DOI 10.1182/blood-2012-04-426247.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2012 by The American Society of Hematology
2428 BLOOD, 20 SEPTEMBER 2012
䡠
VOLUME 120, NUMBER 12For personal use only.
at CALIFORNIA INST TECH/MILLIKAN LIBRARY on October 25, 2012.
bloodjournal.hematologylibrary.org
However, little is known about the physiologic function of the Bach1/HO-1 pathway and its potential role in disease pathology.
In this study, we examine whether Bach1 plays a role in autoimmune disease development and normal immune function. We report here that disruption of Bach1 alone results in partial protection from the development of autoimmune diseases with the use of experimental autoimmune encephalomyelitis (EAE), which is the murine model of human multiple sclerosis (MS).14 This
phenotype is accompanied by defective peripheral T-cell responses in mice. However, deleting Bach1 does not appear to affect T-cell function intrinsically but rather impaired the development of certain APC subsets, specifically macrophages and DCs. We performed genomewide analyses and identified a panel of Bach1 target genes that might be important for APC development. Of these genes, HO-1 was highly up-regulated on deleting Bach1 in APCs. To determine the significance of HO-1 up-regulation, we generated Bach1⫺/⫺HO-1⫺/⫺ mice. Unexpectedly, these animals were nearly depleted of macrophages and had lower numbers of DCs compared with Bach1⫺/⫺ mice, indicating that the Bach1/ HO-1 pathway is a key event for APC development. This pheno-type of the HO-1⫺/⫺Bach1⫺/⫺ mice was accompanied by a significant decrease in the numbers of upstream APC progenitor cells, including CMP-Flk2⫹, but an increase in hematopoietic stem-progenitor cell (HSPC) numbers, suggesting a developmental block in the progression of HSPCs to CMP-Flk2⫹ and subse-quently APCs. Collectively, we identified the CNC member Bach1 as a new player in regulating steady state myelopoiesis, normal immune function, and autoimmune disease development, expand-ing the understandexpand-ing of CNC-Maf biology. Our data indicate that the Bach1/HO-1 pathway provides a key signaling event for the development of stem cells into APCs.
Methods
Materials and flow cytometry
Antibodies for flow cytometry were purchased from eBioscience, BioLeg-end, or Santa Cruz Biotechnology. Flow cytometric analysis was performed with FACSCalibur (BD Bioscience) or MACSQuant (Miltenyi Biotec). The FloJo Version 9.3.2 software (TreeStar) was used for data analyses. GM-CSF and M-CSF were purchased from R&D Systems. PolyI:C was obtained from InvivoGene. Ovalbumin/complete Freund adjuvant (OVA/ CFA) emulsions, MOG35-55/CFA emulsions, and pertussis toxin (PT) were
purchased from Hooke Laboratories. Cytokine ELISA kits were purchased from eBioscience or BioLegend. OVA protein was purchased from Sigma-Aldrich.
Generation of Bach1ⴚ/ⴚand HO-1ⴚ/ⴚBach1ⴚ/ⴚmice
C57bl/6 Bach1⫺/⫺mice were rederived at the animal facility at California Institute of Technology from embryos obtained from the Igarashi labora-tory.14The progeny were further mated to in-house C57bl/6 animals. The
Bach1⫹/⫺pups were bred to obtain wild-type (WT) and C57bl/6 Bach1⫺/⫺ animals, and the respective genotypes were mated to generate experimental WT and Bach1⫺/⫺mice. FVB HO-1⫹/⫺mice31were purchased from The
Jackson Laboratory and crossed with C57bl/6 Bach1⫺/⫺mice. HO-1⫹/⫺ Bach1⫹/⫺ mice were bred, and HO-1⫹/⫺Bach1⫺/⫺animals were subse-quently mated to generate HO-1⫺/⫺Bach1⫺/⫺animals.
BM reconstitution
C57bl/6 recipient animals were irradiated with 12 gray (1200 rad) of cesium. Ten million WT or Bach1⫺/⫺BM cells (BMCs) were injected into the lethally irradiated animals. Six to 8 weeks after reconstitution, the recipient mice were killed or immunized.
ELISAs
ELISAs for T-cell cytokines were performed as described by the manufac-turer. For the MOG35-55–specific IgG ELISA (Bethyl Laboratories), MOG35-55
(2g/mL) peptide was coated onto a 96-well plate overnight. Serially diluted sera were loaded on the plate and quantified according to the protocol described by the manufacturer (Bethyl Laboratories; catalog no. E90-131).
OVA immunization and T-cell recall experiments
Mice were immunized with 200g of OVA protein emulsified in 200 g of CFA subcutaneously. One month after immunization, the animals were boosted with 100g of OVA emulsified in incomplete Freund adjuvant (IFA) subcutaneously. Two weeks after boosting, the spleen was harvested, and red blood cells were lysed. Splenocytes were then cultured in cell media (Hyclone RPMI containingL-glutamine, 10% FBS, penicillin and strepto-mycin, 50M -mercaptoethanol) at a density of 1 million cells per milliliter in 12- or 24-well plates. Cells were treated with 10g/mL OVA.
EAE induction and immune cell infiltration analysis
EAE was induced by subcutaneous injection with emulsions that contained 200g of MOG35-55 and 400g of CFA and 600 ng of PT. The same
amount of PT was injected 22-26 hours later. The following disease scoring system was used: 0.5⫽ partial limp tail, 1 ⫽ complete limp tail, 2 ⫽ wob-bly walk, 2.5⫽ 1-leg paralysis, 3 ⫽ 2-leg paralysis, 3.5 ⫽ 3-leg paralysis, 4⫽ moribund, and 5 ⫽ death. One month after EAE induction, total spinal cord was harvested, stained with fluorochrome-conjugated antibodies, and subjected to flow cytometric analyses to quantify the ratio of immune cellular infiltrates. FACS plots were forward and side scatter gated for hematopoietic cells.
Generation of BM-DCs and BMMs
Cells harvested from the BM were plated in medium that contained 20 ng/mL GM-CSF. On day 4, GM-CSF–containing medium was replen-ished. Three days later, all the surviving cells were harvested and subjected to flow cytometric analyses. To generate BM-derived macrophages (BMMs), BM cells were cultured in media (Hyclone DMEM with 10% fetal bovine serum plus penicillin/streptomycin) supplemented with 10 ng/mL M-CSF. Six days later, the adherent macrophages were replenished with new media without M-CSF, and experiments were performed.
Microarray experiments
RNAs from WT and Bach1⫺/⫺BMMs were submitted to University of California San Francisco Microarray Core Facility for gene expression analyses. Briefly, Agilent 4⫻ 44K GE Mm arrays were used as platforms. Sample preparation, labeling, and array hybridizations were performed according to standard protocols from the University of California San Francisco Shared Microarray Core Facilities. Arrays were scanned with the Agilent microarray scanner, and raw signal intensities were obtained with Feature Extraction version 10.1 software (Agilent). All microarray data are available at ArrayExpress under accession no. E-MEXP-3674.
Statistical analyses
All P values were obtained with Student t tests (unpaired 2-tailed with 95% confidence interval) except for Figure 1A-B, in which 2-way ANOVA was used.
Results
Deletion of Bach1 in mice offers partial protection from development of EAE
As a test of the role of Bach1 in immunity we examined its role in EAE pathogenesis. We induced EAE in WT and Bach1⫺/⫺mice14
in CFA and PT. Standardized clinical disease scoring was used to quantify the severity of paralysis, with higher numbers indicating more severe symptoms. In contrast to Nrf2⫺/⫺ mice,34which get
severe autoimmune disease, Bach1⫺/⫺ mice were partially pro-tected from EAE pathogenesis (Figure 1A). Although 40% of tested WT animals developed complete hind leg paralysis and front leg paresis (clinical score, ⱖ 3.5), only 7% of Bach1⫺/⫺ mice developed such severe EAE symptom (Figure 1B). Of the animals tested, 91% of WT animals developed partial hind leg paralysis (clinical score, 2.5), whereas only 64% of Bach1⫺/⫺ animals exhibited this symptom. Of note, similar percentages of WT and Bach1⫺/⫺animals (100% WT and 96% Bach1⫺/⫺) developed mild EAE symptoms, marked by partial limp tail (clinical score, 0.5). Collectively, these data indicate that Bach1 does not alter the incidence of EAE development but affects the severity of the disease. Consistent with this, the decrease in EAE severity corresponded to less weight loss in Bach1⫺/⫺animals (Figure 1C); we started measuring the weight of the animals after 12 days of EAE induction when signs of disease became noticeable. More-over, Bach1⫺/⫺ mice were less capable of producing antibodies against MOG35-55after immunization (Figure 1D), indicative of a
reduced immune response in these mice.
The pathogenesis of EAE initiates with immune cell infiltration into the CNS. The lower inflammatory condition (Figure 1D) in Bach1⫺/⫺ mice suggested that these animals might be protected from EAE as a result of reduced penetration of immune cells into
the CNS. To assess this, we harvested spinal cord from diseased animals and used flow cytometry to quantify the percentage of infiltrating hematopoietic cells. Consistent with the lower disease severity, fewer APCs (CD45⫹ CD11b⫹MHC II⫹) were found in the CNS of Bach1⫺/⫺ EAE animals (Figure 2A). This was a consequence of both less infiltration of peripheral DCs (Figure 2B) and fewer resident CNS microglia (Figure 2C), which proliferate on EAE development.35In parallel to lower APCs, fewer
infiltrat-ing T and B cells were observed in the CNS of these animals (Figure 2D-F). Thus, during pathogenesis of EAE, ablation of Bach1 partially protects the CNS from immune cell infiltration and disease development.
Bach1 indirectly regulates T cell–mediated immune responses through controlling the steady state development of APCs Our finding that deletion of Bach1 lowered infiltration of immune cell into the CNS during EAE pathogenesis, which is a T cell– mediated disease, provided the impetus for us to examine whether Bach1 regulates T-cell effector responses in the periphery. To test this, we immunized WT and Bach1⫺/⫺mice with OVA, harvested the splenocytes, and restimulated the cultured splenocytes with vehicle or OVA. Indeed, we observed that OVA stimulation of the Bach1⫺/⫺splenocytes induced activation of fewer T cells (Figure 3A) accompanied by impaired T-cell proliferation (Figure 3B) and expansion (Figure 3C). Consistently, we detected in activated Figure 1. Bach1-deficient mice exhibit decreased severity in the autoimmune disease model EAE. WT-type or Bach1⫺/⫺mice were immunized subcutaneously with MOG35-55emulsified in CFA and injected with PT. (A) Bach1⫺/⫺mice had decreased severity of paralysis in the EAE model. After induction of EAE, clinical disease scores were
measured. One of 3 independent experiments is shown; each experiment containedⱖ 7 animals per genotype. Two-way ANOVA was used for P value. (B) Bach1⫺/⫺mice were protected from severe paralysis after EAE induction. The graph shows the percentage of animals that reach the corresponding clinical score. The data were generated with ⬎ 40 WT animals and Bach1⫺/⫺animals. (C) Bach1⫺/⫺mice were less sick after EAE induction. The weights of the mice were taken at the indicated times after EAE induction
and expressed as fraction weight loss. Two-way ANOVA, P⬍ .001 (WT N ⫽ 28; Bach1⫺/⫺N⫽ 29). (D) Bach1⫺/⫺animals produced lower amount of IgG antibodies against MOG35-55. Serum from mice (N⫽ 10) before and 2 weeks after EAE induction were subjected to ELISA analysis to quantify the relative amount of MOG35-55–specific antibody
produced, expressed as OD450 nm in the plot. All graphs are represented with SEM.
2430 SO et al For personal use only. BLOOD, 20 SEPTEMBER 2012
䡠
VOLUME 120, NUMBER 12at CALIFORNIA INST TECH/MILLIKAN LIBRARY on October 25, 2012.
bloodjournal.hematologylibrary.org
splenocytes lower amounts of IL-2, IFN␥, IL-13, and IL-17A cytokines (Figure 3D-H), which are produced by Th1, Th2, and Th17. These data indicate that deletion of Bach1 lowers general CD4⫹T-cell responses, including those of Th1, Th2, and Th17.
To delineate whether Bach1-mediated T-cell response is intrin-sic to the hematopoietic compartment, WT and Bach1⫺/⫺BMCs were reconstituted into lethally irradiated C57bl/6 recipient mice, which were then immunized with OVA. Indeed, fewer CD4⫹ T cells were activated (Figure 4A; supplemental Figure 1A, available on the Blood Web site; see the Supplemental Materials link at the top of the online article) and lower amounts of T-cell cytokines (Figure 4B) were produced from the splenocytes har-vested from animals reconstituted with Bach1⫺/⫺ BMCs. Impor-tantly, treatment of WT and Bach1⫺/⫺splenocytes with the direct T cell–specific activation ligand ConA led to similar proliferation of T cells (Figure 4C) and production of equal amounts of cytokines (Figure 4D). In addition, CD4⫹ T cells purified from OVA-immunized WT or Bach1⫺/⫺mice were similarly responsive to OVA activation when cocultured with naive total WT spleno-cytes, which served as a source of APCs. This was indicated by the percentage of CD4⫹CD25⫹-activated T cells and IL-2 secretion (Figure 4E-F). Collectively, these data indicated that Bach1 regulates T-cell responses through its function within the hemato-poietic compartment but does not function intrinsically in T cells for activation. This suggested that Bach1 regulates upstream events to modulate T-cell activation and responses. To further determine
the mechanism by which Bach1 regulates T-cell immunity, we determined whether disruption of Bach1 had any effect on APCs. Interestingly, although B-cell numbers were similar in Bach1⫺/⫺ mice (Figure 4G; supplemental Figure 1B), fewer APCs (macro-phages and DCs; Figure 4H-I; supplemental Figure 1C-D) were observed in these animals. Hence, Bach1 appears to be needed for production of normal numbers of specific types of APCs and thus regulates T-cell responses indirectly.
To more precisely characterize the numbers of various cell types in the knockout animals, splenocytes from naive WT and Bach1⫺/⫺ mice were subjected to flow cytometric analyses. The total number of splenic leukocytes between the 2 groups of animals was similar (supplemental Figure 1E). Although the percentage of T cell (CD3⑀⫹), B cells (CD19⫹), natural killer cells (NK1.1⫹CD49b⫹), and erythrocytes (Ter119⫹; Figure 5A-D; supplemental Figure 2A-D) were similar between the 2 groups, the fractions of macrophages (MHC II⫹ CD11c⫺ CD11b⫹ F4/80⫹; Figure 5E; supplemental Figure 2E) and CD11c⫹DCs (Figure 5F; supplemen-tal Figure 2F) were significantly lower in the Bach1⫺/⫺animals. Furthermore, the populations of macrophage-committed (CD11b⫹ F4/80⫹ CD115⫹; Figure 5G; supplemental Figure 2G) and DC-committed precursors5,6were lower in the BM compartment of the
mutant animals (Figure 5H; supplemental Figure 2H).
To determine whether the decrease in macrophage and DC populations in Bach1⫺/⫺ mice was responsible for the lowered T-cell responses (Figure 4), we cocultured splenocytes OTII CD4⫹ Figure 2. Bach1ⴚ/ⴚEAE mice display less inflammation in the CNS than WT animals. Spinal cords of mice were harvested 1 month after EAE induction, and immune cell
infiltrates were quantified by FACS. The left graph represents the FACS plots after forward scatter and side scatter gating, and the right plot shows the compiled data of the relative percentage of corresponding immune cells infiltrated into the spinal cord of the EAE animals. (A) Lower APCs (CD45⫹CD11b⫹MHC II⫹) in Bach1⫺/⫺mice induced with EAE. Displayed FACS plots are CD45⫹gated. (B) DCs were distinguished as CD45⫹CD11c⫹GR-1⫺cells. Displayed FACS plots are GR-1⫺gated. (C) Microglia population was marked as CD45intCD11b⫹population. (D) Infiltration of pan T cells in the spinal cord were identified as CD45⫹CD3⑀⫹cells. (E) Th infiltrates were distinguished as
CD3e⫹CD4⫹cells (cell population in pink box). (F) B cells in the spinal cord were identified as CD45⫹CD19⫹cells (cell population in pink box). Bolded letters of the surface markers denote that the plot shown are after gating to that particular marker. Each group containsⱖ 15 animals. Unpaired 2-tail Student t test performed for all the panels to obtain P value. The horizontal bars on each graph represents the average percentage.
T cells in the presence of OVA. A given number of Bach1⫺/⫺ splenocytes were less able than WT cells to induce an OVA OTII CD4⫹ T-cell response, which is consistent with Bach1⫺/⫺ mice having fewer APCs (Figure 5I). Thus, our data indicate that Bach1 regulates the steady state level of specific populations of APCs, limiting the magnitude of T-cell responses.
Bach1 regulates development of APCs in a cell-autonomous manner
To investigate whether Bach1 functions within the hematopoietic compartment to activate APC development, we reconstituted lethally irradiated C57bl/6 WT recipient mice with WT or Bach1⫺/⫺ BMCs. The animals reconstituted with Bach1⫺/⫺BMCs exhibited fewer macrophages and DCs in their spleens than animals that received WT BMCs (Figure 5J-K). To test whether Bach1 func-tions in other nonhematopoietic compartments to mediate DC development, WT and Bach1⫺/⫺ lethally irradiated mice were reconstituted with WT BMCs. Both recipient groups displayed a similar percentage of splenic macrophages and DCs (data not shown). Thus, Bach1 influences the development of these APCs by functioning within the hematopoietic compartment and not through other nonhematopoietic tissues. To examine whether Bach1 modu-lates APC development in a cell-autonomous manner, BMCs were isolated and treated with GM-CSF to generate DCs cells in vitro.36
Similar to our in vivo finding, BMCs harvested from Bach1⫺/⫺ mice produced⬍ 50% of the DCs generated by WT cells (Figure 5L). These in vitro experiments combined with our in vivo analyses imply that Bach1 is directly involved in the development of a subset of APCs through a cell-autonomous mechanism.
Identification of Bach1 targets
To provide insights into the mechanism and molecular pathways by which Bach1 regulates APC development, we performed whole-genome gene expression microarray to identify gene targets regulated by Bach1 in BMMs. With the use of this unbiased approach, we identified ⬎ 300 genes differentially expressed in WT versus Bach1⫺/⫺BMMs (Figure 6A; supplemental Figure 3). We validated by quantitative PCR a panel of genes up-regulated on deletion of Bach1⫺/⫺(Figure 6B). In our microarrays, we found that the known direct Bach1 target HO-1 was the most highly up-regulated in Bach1⫺/⫺ BMMs.14,28-31 In addition, we also
isolated previously unknown Bach1 targets, including Clec4n and Fl3a1, as well as Slc40a1/Fpn1. Slc40a1/Fpn1 was previously found to be concomitantly up-regulated with HO-1 after eryth-rophagocytosis.37Because HO-1 was identified in our microarrays
as the most affected by Bach1 ablation, we next examined whether HO-1 could also be regulated by Bach1 in total primary BMCs. Indeed, we found that Bach1⫺/⫺ BMCs expressed significantly Figure 3. Defective Th response on ablation of Bach1. WT or Bach1⫺/⫺mice were immunized subcutaneously with 100g of OVA/CFA. One month after immunization, the animals were boosted with 100g in IFA. Splenocytes were isolated, equal numbers were plated and cultured with OVA (10 g/mL). (A) Bach1⫺/⫺splenocytes are defective in
T-cell activation. After 2 days of OVA treatment, splenocytes were subjected to flow cytometric analysis to determine the percentage of T cells that expressed the activation marker (CD25) after OVA induction. The graph shows the average percentage of CD3⑀⫹CD25⫹population in the splenocytes. P⬍ .05. KO indicates knockout. (B) Bach1⫺/⫺
splenocytes are defective in antigen-specific–stimulated proliferation. Splenocytes were pulsed with tritiated (3H) thymidine for 16-24 hours. The incorporated radiolabeled
thymidine in the cells was quantified in scintillation fluid and measured as counts per minute (CPM). The time on the x-axis indicates the hours of OVA stimulation, which also includes the3H thymidine pulse time. Two-way ANOVA of WT OVA versus Bach1⫺/⫺OVA, P⬍ .001. (C) Impaired OVA-induced T-cell expansion on deletion of Bach1.
Splenocytes were cultured for 60 hours, and flow cytometry was used to quantify the percentage of CD3⑀ T cells. (D-H) Defective production of T-cell cytokines on OVA restimulation in Bach1⫺/⫺splenocytes. After OVA restimulation of culture splenocytes for 3 days, ELISAs were performed with the supernatant fluid to measure cytokine production. Three independent experiments were performed, and each experiment contained 4-12 mice per genotype. WT OVA versus Bach1⫺/⫺OVA, P⬍ .05. All plots are expressed with SEM.
2432 SO et al For personal use only. BLOOD, 20 SEPTEMBER 2012
䡠
VOLUME 120, NUMBER 12at CALIFORNIA INST TECH/MILLIKAN LIBRARY on October 25, 2012.
bloodjournal.hematologylibrary.org
higher levels of HO-1 transcript (Figure 6C). Collectively, we identified a panel of Bach1 target genes that may be involved in Bach1-mediated APC development.
Deletion of HO-1 exacerbates the impaired APC development observed in Bach1ⴚ/ⴚmice
To investigate the mechanism by which Bach1 controls APC development, we examined the function of HO-1 up-regulated expression in the context of Bach1 ablation in hematopoietic development. We chose to study HO-1 because it has been previously identified as a direct target of Bach114,28,29 and was
identified as the most affected gene in our microarrays by Bach1 deletion (Figure 6A-B). To this end, we generated HO-1⫺/⫺ Bach1⫺/⫺ mice by breeding Bach1⫺/⫺ with HO-1⫹/⫺ mice31and
intercrossing their subsequent progeny. Breeding of HO-1⫹/⫺ Bach1⫹/⫺ parent pairs did not produce any HO-1⫺/⫺Bach1⫺/⫺ animals (36 pups genotyped), and breeding of HO-1⫹/⫺Bach1⫺/⫺ pairs resulted in⬍ 2% of pups being HO-1⫺/⫺Bach1⫺/⫺, which is much lower than the expected 25% Mendelian distribution (chi square P⬍ .001). In the BM compartment, we detected almost no
macrophages in the HO-1⫺/⫺Bach1⫺/⫺ mice (Figure 6D). The compound HO-1⫺/⫺Bach1⫺/⫺ mice had even fewer DCs than Bach1⫺/⫺animals (Figure 6E). It was reported that the generation of HO-1⫺/⫺ mice was more successful in FVB than the C57bl/6 background.38Thus, we attempted to generate FVB HO-1⫺/⫺mice
through breeding heterozygotes; however, our breeding of HO-1⫹/⫺ pairs did not produce any HO-1⫺/⫺pups (⬎ 90 pups genotyped), precluding us from being able to examine APC development in mice with only HO-1 deletion. Nevertheless, our data indicate that the Bach1/HO-1 pathway is essential for macrophage development in the BM and necessary for optimal development of DCs.
In addition, we also assessed whether the Bach1/HO-1 pathway regulates the development of other hematopoietic cells. We found that the HO-1⫺/⫺Bach1⫺/⫺ mice had similar numbers of T-cells (CD4⫹CD3⑀⫹, CD8␣⫹CD3⑀⫹), natural killer cells (NK1.1⫹GR-1⫺), B cells (CD19⫹GR-1⫺), and GR-1⫹ myeloid cells as the Bach1⫺/⫺ mice (supplemental Figure 4), which have normal development of these cells compared with WT animals as described above (Figure 5). This indicates that the Bach1/HO-1 pathway regulates APC development without affecting these immune cells.
Figure 4. Impaired T-cell responses in C57bl/6 recipient animals reconstituted with BMCs from Bach1ⴚ/ⴚdonors. Lethally irradiated C57bl/6 recipients reconstituted
with BMCs from WT or Bach1⫺/⫺donors were immunized with OVA/CFA, and splenocytes were cultured in the presence or absence of OVA. After 3 days in culture, splenocytes were subjected to (A) flow cytometric analyses (see also supplemental Figure 1A) to quantify the percentage of CD4⫹cells that expressed the CD25 T-cell activation marker and (B) the supernatant fluids were examined for the amount of IFN␦ with the use of ELISAs. Student t test P ⬍ .05 for the OVA-treated samples. (C) Total splenocytes from WT or Bach1⫺/⫺animals were stimulated with concanavalin A (ConA) and pulsed with tritiated thymidine for the last 16-22 hours. (D) The amount of IL-2 secreted by T cells in total splenocytes after stimulation with ConA was determined by ELISA. (E-F) WT and Bach1⫺/⫺mice were immunized with OVA/CFA and boosted with OVA/IFA. Splenic CD4⫹T cells from these animals were purified 2 weeks later and cocultured with naive total WT splenocytes in the absence or presence of OVA for 72 hours. The percentage of activated CD4⫹T cells (CD4⫹CD25⫹) was measured by flow cytometry, (E) and the amount of secreted IL-2 was quantified by ELISA (F). Splenocytes from animals that had undergone immunization with OVA/CFA were subjected to flow cytometric analyses to examine the percentage of B cells (G), macrophages (H), and DCs (I). See also supplemental Figure 1B, C, and D for flow cytometric plots for panels G, H, and I, respectively. The data represent averages ofⱖ 3 mice per group.
The Bach1/HO-1 pathway influences the development of APC precursors and multipotent myeloid progenitors
To further probe the mechanism by which Bach1/HO-1 pathway orchestrates APC development, we assessed whether Bach1/HO-1 regulates APC precursor cells (see Figure 7A). We examined and found that ablating Bach1 and HO-1 resulted in impaired develop-ment of monocytes (Figure 7B), precursors of macrophages.5,6The
HO-1⫺/⫺Bach1⫺/⫺mice were also defective in developing precur-sors of the DC-lineage. The pre-cDCs (Figure 7C) and the more upstream common DC progenitors (CDPs; Figure 7D) were both reduced in HO-1⫺/⫺Bach1⫺/⫺ mice. Monocytes and CDPs are generated from the macrophage-DC precursor (MDPs; Figure 7A).7-9Further upstream of MDPs in APC development are Flk2⫹
CMPs (CMP-Flk2⫹) that have multipotent myeloid-forming poten-tial.39 Strikingly, HO-1⫺/⫺Bach1⫺/⫺ mice had fewer numbers of
both MDPs and CMP-Flk2⫹ (Figure 7E-F), indicating that the Bach1/HO-1 pathway controls the production of APC by control-ling the development of multipotent myeloid progenitors. In contrast, the HO-1⫺/⫺Bach1⫺/⫺ mice have an increase in their fraction of Lin⫺ cKit⫹ Sca1⫹ pluripotent HSPCs (Figure 7G). Further analyses indicated that these mice had an elevated percent-age of MPP and ST-HSCs (Figure 7H). Collectively, our data suggest that ablation of the Bach1/HO-1 signaling pathway results
in a developmental block from the progression of MPP/ST-HSCs to CMP-Flk2⫹ and subsequently APCs. Thus, we conclude that the Bach1/HO-1 pathway is an important event for committing pluripo-tent stem-progenitor cells to develop into APCs.
Discussion
Here, we found that optimal developmental progression from pluripotent stem cells to macrophages and DCs requires Bach1/ HO-1 signaling. First, we found that Bach1, a member of the CNC protein family, contributes to the development of APCs, specifi-cally macrophages and DCs. The impaired APC development in Bach1-deficient mice was accompanied by defects in downstream Th responses, indicating the functional significance of Bach1 in immune responses. Importantly, and consistent with a dampened T-cell response, ablation of Bach1 in mice resulted in partial protection of these animals from development of the T cell– mediated autoimmune disorder EAE. Thus, we have defined a new player in immune function and shown that Bach1 influences hematopoiesis and immunity in a manner that affects the develop-ment of autoimmune disease. We next identified a panel of Bach1 target genes and examined whether the most highly regulated gene, Figure 5. Bach1 regulates steady state development of macrophages and DCs through its intrinsic function in the BM compartment. White blood cells (WBCs)
harvested from splenocytes of age-matched adult animals were subjected to FACS analyses to quantify the percentage population of (A) T lymphocytes (CD3⑀⫹), (B) non–plasma B cells (CD19⫹), (C) natural killer cells (NK1.1⫹, CD49b⫹), (D) erythrocytes (Ter-119⫹), (E) monocyte/macrophages (F4/80⫹CD11b⫹CD11c⫺MHC II⫹), and (F) DCs (CD11c⫹MHC II⫹). (G-H) Bach1 regulates the development of DC-precursors and monocytes. WT and Bach1⫺/⫺BM cells were subjected to flow cytometric analyses to quantify the percentage of DC-precursors (G) and monocytes (H). LIN contains a cocktail of Ter-119, CD11b, B220, GR-1, and CD3⑀. (I) Equal numbers of splenocytes from naive WT or Bach1⫺/⫺mice were cocultured with purified OTII CD4⫹T cells in the absence or presence of OVA for 72 hours. The amount of secreted IL-2 was quantified by ELISA. WT versus Bach1⫺/⫺OVA-treated samples: P⬍ .05. (J) Macrophages and (K) DCs (right panel) in the BM of lethally irradiated C57bl/6 recipients reconstituted with BMCs from WT or Bach1⫺/⫺donors. (L) Bach1 influences the development of BM cells into DCs in vitro. Equal numbers of WT and Bach1⫺/⫺BMCs were cultured with 20 ng/mL GM-CSF. Total number of DCs (CD11c⫹MHC II⫹) generated at day 7 was calculated by cell counting and flow cytometric analyses. All data represent experiments containingⱖ 3 animals per genotype. The graphs are plotted with SEM.
2434 SO et al For personal use only. BLOOD, 20 SEPTEMBER 2012
䡠
VOLUME 120, NUMBER 12at CALIFORNIA INST TECH/MILLIKAN LIBRARY on October 25, 2012.
bloodjournal.hematologylibrary.org
HO-1, serves as an effector in APC development. Through these analyses, we found that Bach1/HO-1 signaling is critical for DC and macrophage development, indicating that this pathway is important for maintaining steady state hematopoiesis in addition to the previously predicted heme homeostasis.33 Mechanistically,
because HO-1⫺/⫺Bach1⫺/⫺double mutants had a decreased popula-tion of early APC progenitor cells (CMP-Flk2⫹) accompanied by an increased fraction of the further upstream HSPCs, it suggests that these mice exhibit a developmental block in the progression from HSPCs to CMP-Flk2⫹ and subsequently APCs. Thus, we infer that the Bach1/HO-1 pathway regulates a key signaling event for committing stem cells to develop into APCs.
Advances have been made toward the identification of precur-sor cells that are capable of developing into APCs. It has been shown that DCs are generated from Lin⫺Flt3⫹CD115⫹CD117 (cKit)lo
CDP cells.6 Macrophages and DCs are derived from common
precursors coined MDP.7-9 MDPs are probably derived from
upstream CMP-Flk2⫹ cells, which can be differentiated into myeloid cells but are restricted from forming erythroid cells.39
CMP-Flk2⫹cells themselves are produced from pluripotent HSCs. The biologic program that directs CDPs, MDPs, and CMP-Flk2⫹ development and the signaling pathways that commit HSCs to differentiate into CMP-Flk2⫹ have not been well defined. We report here that Bach1/HO-1 signaling contributes importantly to the generation of CDPs, MDPs, and CMP-Flk2⫹. Further, a decrease in the number of these cells in HO-1⫺/⫺Bach1⫺/⫺mice was accompanied by an increase in HSC population, suggesting that Bach1/HO-1 probably provides a positive signal for either committing HSCs toward CMP-Flk2⫹lineage or negative signal to inhibit HSCs from differentiation.
CNC proteins and their interaction partners small Mafs are widely studied in part because of their potentially extensive
therapeutic interest, including in MS, aging, respiratory diseases, and cancer. Although advances toward understanding the involve-ment of Bach1 in cytoprotection during liver and myocardial ischemia have been made,40,41 the biologic role of the CNC
member Bach1 is still largely unknown. Here, we show that, in addition to Bach1-mediated normal programming of APC develop-ment, it plays a role in generating optimal immunity and autoimmune-related pathology. Indeed, Bach1⫺/⫺ mice are im-paired in launching a T cell–mediated response, which is probably attributed to their abnormal APC development because Bach1 does not appear to function intrinsically in T cells. Of importance, the deletion of Bach1⫺/⫺ partially protects animals from developing EAE, connecting Bach1 function to the severity of autoimmune diseases. It has been shown that Nrf2, a relative of Bach 1, inhibits the maturation of DCs in vitro42 and protects humans from MS
development.25,26 Potentially, Nrf2 and Bach1 might serve as
counterparts in the CNC/Maf regulatory network, regulating the homeostatic balance of DC development and MS pathogenesis. Moreover, it is important to highlight that deletion of Bach1 did not completely ablate APC development, suggesting that other CNC and/or small Maf subunits might have compensatory or redundant functions with Bach1. Potentially, up-regulation of HO-1 might protect APC development in Bach1⫺/⫺mice because deleting HO-1 in these animals exacerbated the decrease in DC and macrophage numbers. Alternatively, up-regulation of other target genes, such as Spi-C, which has been found to regulate the genesis of red pulp macrophages,43might protect APC development in Bach1⫺/⫺mice.
During the preparation of this manuscript, it was shown that ablating HO-1 resulted in diminished number of macrophages in the spleen.44 In that study, it was found that deleting HO-1 in
BM-derived macrophages resulted in toxicity of these cells on red Figure 6. HO-1 ablation exacerbates impaired APC development in Bach1ⴚ/ⴚmice. (A) RNA from WT and Bach1⫺/⫺BMMs were harvested and reverse transcribed, and cDNA was subjected for microarray analyses. Genes highlighted in red or green represent those up- or down-regulated, respectively, in Bach1⫺/⫺BMMs. See also supplemental Figure 3. (B) Quantitative PCR analyses to verify Bach1 target genes identified in the microarrays. WT and Bach1⫺/⫺BMMs were harvested for gene analyses. Relative transcript levels were normalized to the house keeping gene Rpl19. (C) Bach1 regulates HO-1 in BM cells. Transcript level of HO-1 was assessed from samples harvested from WT and Bach1⫺/⫺BMCs from 3 separate animals. (D-E) BM cells were isolated from mice of the indicated genotype and subjected to flow cytometric analyses to measure the percentage of (D) macrophages and (E) DCs. Each dot represents an individual animal. Red graph contains 3 HO-1⫹/⫹Bach1⫺/⫺and 5 HO-1⫹/⫺Bach1⫺/⫺mice. Blue represents 3 HO-1⫹/⫺Bach1⫺/⫺mice. All graphs are plotted with SEM. The horizontal bars on each graph represents the average percentage.
blood cell phagocytosis, probably as a result of heme-overloaded toxicity. In our study, we found that the impaired APC development is also associated with the generation of their progenitors. Thus, it appears that HO-1 controls the number of APCs at a stage before as well as after development into mature APCs. Collectively, we also identified a panel of previously unknown Bach1 targets. It will be interesting in the future to examine whether those genes also play a role in APC development. Potentially, Bach1 may regulate distinct or overlapping subsets of those genes for DC versus macrophage development.
Acknowledgments
The authors thank Dr Kazuhiko Igarashi for sharing the Bach1⫺/⫺ mice and are grateful to Alex Balazs and Michael Bethune for helpful critiques of the manuscript.
This work was supported by the National Institutes of Health (grants 5P01CA132681-02 and 5R01AI093531-02 to D.B. and grant F32 CA139883 to A.Y.-L.S.).
Authorship
Contribution: A.Y.-L.S., Y.G.-F., A. Minisandram, A. Martin, and K.T. performed the experiments; A.Y.-L.S., M.B., and D.B. con-ceived the study; A.Y.-L.S., Y.G.-F., A. Mehta, A.Y.-L.S., K.T., M.B., and D.B. provided crucial reagents; A.Y.-L.S. and D.B. wrote the manuscript; and all authors provided input on the manuscript.
Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.
Correspondence: David Baltimore, California Institute of Tech-nology, 1200 East California Boulevard MC 147-75, Pasadena, CA 90227; e-mail: [email protected].
References
1. Liu K, Nussenzweig MC. Origin and development of dendritic cells. Immunolog Rev. 2010;234(1): 45-54.
2. Liu K, Victora GD, Schwickert TA, et al. In vivo analysis of dendritic cell development and ho-meostasis. Science. 2009;324(5925):392-397. 3. Naik SH, Metcalf D, van Nieuwenhuijze A, et al.
Intrasplenic steady-state dendritic cell precursors that are distinct from monocytes. Nat Immunol. 2006;7(6):663-671.
4. Naik SH, O’Keeffe M, Proietto A, Shortman HH, Wu L. CD8⫹, CD8-, and plasmacytoid dendritic cell generation in vitro using flt3 ligand. Methods Mol Biol. 2010;595:167-176.
5. Naik SH, Sathe P, Park HY, et al. Development of plasmacytoid and conventional dendritic cell sub-types from single precursor cells derived in vitro and in vivo. Nat Immunol. 2007;8(11):1217-1226. 6. Onai N, Obata-Onai A, Schmid MA, Ohteki T,
Jarrossay D, Manz MG. Identification of clono-genic common Flt3⫹M-CSFR⫹ plasmacytoid
and conventional dendritic cell progenitors in mouse bone marrow. Nat Immunol. 2007;8(11): 1207-1216.
7. Fogg DK, Sibon C, Miled C, et al. A clonogenic bone marrow progenitor specific for macro-phages and dendritic cells. Science. 2006; 311(5757):83-87.
8. Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, Ley K. Development of monocytes, macrophages, and dendritic cells. Science. 2010; 327(5966):656-661.
Figure 7. BM cells were harvested from adult mice and subjected to flow cytometric analyses. (A) Development of macrophages and DCs from HSCs. HSCs give rise to
CMP-Flk2⫹. Macrophage-DC-restricted progenitors (MDPs) can generate both macrophages and DCs, whereas common DC progenitors (CDPs) are restricted to formation of DCs. The panels correspond to the following cells: (B) monocytes, (C) pre-cDCs, (D) CDPs, (E) MDPs, (F) CMP-Flk2⫹cells, (G) HSPCs, and (H) multipotent progenitor (MPP) or short-term hematopoietic stem cells (ST-HSCs). Each plot representsⱖ 3 mice. The graphs are plotted with SEM.
2436 SO et al For personal use only. BLOOD, 20 SEPTEMBER 2012
䡠
VOLUME 120, NUMBER 12at CALIFORNIA INST TECH/MILLIKAN LIBRARY on October 25, 2012.
bloodjournal.hematologylibrary.org
9. Waskow C, Liu K, Darrasse-Jeze G, et al. The receptor tyrosine kinase Flt3 is required for den-dritic cell development in peripheral lymphoid tis-sues. Nat Immunol. 2008;9(6):676-683. 10. Sykiotis GP, Bohmann D. Stress-activated
cap‘n’collar transcription factors in aging and hu-man disease. Sci Signal. 2010;3(112):re3. 11. An JH, Blackwell TK. SKN-1 links C. elegans
mesendodermal specification to a conserved oxi-dative stress response. Genes Dev. 2003;17(15): 1882-1893.
12. Bishop NA, Guarente L. Two neurons mediate diet-restriction-induced longevity in C. elegans. Nature. 2007;447(7144):545-549.
13. Motohashi H, O’Connor T, Katsuoka F, Engel JD, Yamamoto M. Integration and diversity of the regulatory network composed of Maf and CNC families of transcription factors. Gene. 2002; 294(1-2):1-12.
14. Sun J, Hoshino H, Takaku K, et al. Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1 gene. EMBO J. 2002;21(19):5216-5224.
15. Blank V. Small Maf proteins in mammalian gene control: mere dimerization partners or dynamic transcriptional regulators? J Mol Biol. 2008; 376(4):913-925.
16. Chan JY, Kwong M, Lu R, et al. Targeted disrup-tion of the ubiquitous CNC-bZIP transcripdisrup-tion fac-tor, Nrf-1, results in anemia and embryonic lethal-ity in mice. EMBO J. 1998;17(6):1779-1787. 17. Shivdasani RA, Rosenblatt MF, Zucker-Franklin D,
et al. Transcription factor NF-E2 is required for plate-let formation independent of the actions of thrombo-poietin/MGDF in megakaryocyte development. Cell. 1995;81(5):695-704.
18. Muto A, Tashiro S, Nakajima O, et al. The tran-scriptional programme of antibody class switch-ing involves the repressor Bach2. Nature. 2004; 429(6991):566-571.
19. Hubbs AF, Benkovic SA, Miller DB, et al. Vacuolar leukoencephalopathy with widespread astroglio-sis in mice lacking transcription factor Nrf2. Am J Pathol. 2007;170(6):2068-2076.
20. Li J, Stein TD, Johnson JA. Genetic dissection of systemic autoimmune disease in Nrf2-deficient mice. Physiol Genomics. 2004;18(3):261-272. 21. Yoh K, Itoh K, Enomoto A, et al. Nrf2-deficient female mice develop lupus-like autoimmune ne-phritis. Kidney Int. 2001;60(4):1343-1353.
22. Derjuga A, Gourley TS, Holm TM, et al. Complex-ity of CNC transcription factors as revealed by gene targeting of the Nrf3 locus. Mol Cell Biol. 2004;24(8):3286-3294.
23. Onodera K, Shavit JA, Motohashi H, Yamamoto M, Engel JD. Perinatal synthetic lethality and hemato-poietic defects in compound mafG::mafK mutant mice. EMBO J. 2000;19(6):1335-1345. 24. Bourquin JP, Subramanian A, Langebrake C,
et al. Identification of distinct molecular pheno-types in acute megakaryoblastic leukemia by gene expression profiling. Proc Natl Acad Sci U S A. 2006;103(9):3339-3344.
25. Kappos L, Gold R, Miller DH, et al. Efficacy and safety of oral fumarate in patients with relapsing-remitting multiple sclerosis: a multicentre, ran-domised, double-blind, placebo-controlled phase IIb study. Lancet. 2008;372(9648):1463-1472. 26. Wilms H, Sievers J, Rickert U, Rostami-Yazdi M,
Mrowietz U, Lucius R. Dimethylfumarate inhibits microglial and astrocytic inflammation by sup-pressing the synthesis of nitric oxide, IL-1beta, TNF-alpha and IL-6 in an in-vitro model of brain inflammation. J Neuroinflammation. 2010;7:30. 27. Ferrando-Miguel R, Cheon MS, Yang JW,
Lubec G. Overexpression of transcription factor BACH1 in fetal Down syndrome brain. J Neural Transm Suppl. 2003;(67):193-205.
28. Kitamuro T, Takahashi K, Ogawa K, et al. Bach1 functions as a hypoxia-inducible repressor for the heme oxygenase-1 gene in human cells. J Biol Chem. 2003;278(11):9125-9133.
29. Sun J, Brand M, Zenke Y, Tashiro S, Groudine M, Igarashi K. Heme regulates the dynamic ex-change of Bach1 and NF-E2-related factors in the Maf transcription factor network. Proc Natl Acad Sci U S A. 2004;101(6):1461-1466.
30. Poss KD, Tonegawa S. Reduced stress defense in heme oxygenase 1-deficient cells. Proc Natl Acad Sci U S A. 1997;94(20):10925-10930. 31. Poss KD, Tonegawa S. Heme oxygenase 1 is
re-quired for mammalian iron reutilization. Proc Natl Acad Sci U S A. 1997;94(20):10919-10924. 32. Chora AA, Fontoura P, Cunha A, et al. Heme
oxygenase-1 and carbon monoxide suppress au-toimmune neuroinflammation. J Clin Invest. 2007; 117(2):438-447.
33. Ogawa K, Sun J, Taketani S, et al. Heme medi-ates derepression of Maf recognition element through direct binding to transcription repressor Bach1. EMBO J. 2001;20(11):2835-2843.
34. Johnson DA, Amirahmadi S, Ward C, Fabry Z, Johnson JA. The absence of the pro-antioxidant transcription factor Nrf2 exacerbates experimen-tal autoimmune encephalomyelitis. Toxicol Sci. 2010;114(2):237-246.
35. Shankaran M, Marino ME, Busch R, et al. Mea-surement of brain microglial proliferation rates in vivo in response to neuroinflammatory stimuli: application to drug discovery. J Neurosci Res. 2007;85(11):2374-2384.
36. Pulendran B, Smith JL, Caspary G, et al. Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc Natl Acad Sci U S A. 1999;96(3):1036-1041.
37. Knutson MD, Vafa MR, Haile DJ, Wessling-Resnick M. Iron loading and eryth-rophagocytosis increase ferroportin 1 (FPN1) ex-pression in J774 macrophages. Blood. 2003; 102(12):4191-4197.
38. Kapturczak MH, Wasserfall C, Brusko T, et al. Heme oxygenase-1 modulates early inflamma-tory responses: evidence from the heme oxygenase-1-deficient mouse. Am J Pathol. 2004;165(3):1045-1053.
39. Nutt SL, Metcalf D, D’Amico A, Polli M, Wu L. Dynamic regulation of PU.1 expression in multi-potent hematopoietic progenitors. J Exp Med. 2005;201(2):221-231.
40. Iida A, Inagaki K, Miyazaki A, Yonemori F, Ito E, Igarashi K. Bach1 deficiency ameliorates hepatic injury in a mouse model. Tohoku J Exp Med. 2009;217(3):223-229.
41. Yano Y, Ozono R, Oishi Y, et al. Genetic ablation of the transcription repressor Bach1 leads to myocardial protection against ischemia/reperfu-sion in mice. Genes Cells. 2006;11(7):791-803. 42. Williams MA, Rangasamy T, Bauer SM, et al.
Dis-ruption of the transcription factor Nrf2 promotes pro-oxidative dendritic cells that stimulate Th2-like immunoresponsiveness on activation by am-bient particulate matter. J Immunol. 2008;181(7): 4545-4559.
43. Kohyama M, Ise W, Edelson BT, et al. Role for Spi-C in the development of red pulp macro-phages and splenic iron homeostasis. Nature. 2009;457(7227):318-321.
44. Kovtunovych G, Eckhaus MA, Ghosh MC, Ollivierre-Wilson H, Rouault TA. Dysfunction of the heme recycling system in heme oxygenase 1-deficient mice: effects on macrophage viability and tissue iron distribution. Blood. 2010;116(26): 6054-6062.
