A transcription factor PU.1 is critical for Ccl22 gene expression in dendritic cells and macrophages

The chemokine CCL22 is predominantly produced by dendritic cells (DCs) and macrophages. CCL22 acts on CCR4-expressing cells including Th2 and Treg. Although a correlation between the CCL22-CCR4 axis and allergic diseases has been established, the mechanism of monocyte lineage-specific Ccl22 gene expression is largely unknown. In the current study, we investigated transcriptional regulation of the Ccl22 gene in DCs and macrophages. Using reporter assays, we identified the critical cis-enhancing elements at 21/−18 and −10/−4 in the Ccl22 promoter. Electrophoretic mobility shift assays proved that transcription factor PU.1 directly binds to the cis-elements. Knockdown of PU.1 markedly decreased Ccl22 expression in bone marrow-derived DCs (BMDCs) and BM macrophages (BMDMs). Chromatin immunoprecipitation assays revealed that PU.1 bound to the Ccl22 promoter in not only BMDCs and BMDMs, but also splenic DCs and peritoneal macrophages. LPS stimulation increased the amount of PU.1 recruited to the promoter, accompanied by upregulation of the Ccl22 mRNA level, which was diminished by Spi1 knockdown. We identified similar cis-elements on the human CCL22 promoter, which were bound with PU.1 in human monocytes. Taken together, these findings indicate that PU.1 transactivates the Ccl22 gene in DCs and macrophages by directly binding to the two elements in the promoter.

PU.1 binding directly at the two proximal sites in the mouse Ccl22 promoter. To identify transcription factor binding to the cis-enhancing elements containing Ets motifs (Site1 at −21/−18 and Site2 at −10/−4), EMSAs were performed using probes described in Fig. 2a. When a FLO-labeled probe containing two sites was mixed with the nuclear proteins extracted from JAWSII cells, several bands appeared on an electrophoretic gel (Fig. 2b lane 2). Among members of the Ets transcription factor family, PU.1/Spi1 especially plays critical roles in differentiation and cell type-specific gene expression of DCs and macrophages. Therefore, we hypothesized that PU.1 transactivates the Ccl22 gene that is apparently expressed in monocytic lineages. To confirm that PU.1 binds to the probe, an anti-PU.1 Ab was added into the mixture of the probe and nuclear extracts. As shown in Fig. 2b lane 3, the most major band disappeared in the presence of an anti-PU.1 Ab, whereas the addition of a non-specific Ab did not affect this major band (lane 4). This result indicates that PU.1 dominantly binds to the Ccl22 minimum promoter region in transcription factors expressed in monocytes. A clearer shift band containing PU.1 was detected in an EMSA, which was carried out by using a recombinant PU.1 protein generated using an in vitro transcription/translation system (Fig. 2c, lanes 10-12, and Supplemental Fig. 1). The specific band was also observed when FLO-labeled probes containing either Site 1 or Site 2 were used (Fig. 2c, lanes 2-4 and [6][7][8]. We then performed an EMSA with competitors to identify the PU.1 binding sites in the probe sequence. The shifted band completely disappeared in the presence of an excess amount of the wild-type (WT) competitors, but not mutant competitors (Fig. 2d). These results suggest that PU.1 is capable of binding to −21/−18 GGAA and −10/−4 TTCTTCT sequences in the mouse Ccl22 promoter. To further elucidate the involvement of PU.1 and the cis-elements in mouse Ccl22 promoter activity, a luciferase assay was carried out using 293 T cells, because this non-hematopoietic cell line, in which PU.1 is not detected, is useful to evaluate the effect of exogenously expressed PU.1. Whereas the luciferase activity driven by the WT promoter was enhanced by co-expression of PU.1, introduction of mutations into both Site 1 and Site 2 completely diminished PU.1/Spi1 knockdown by siRNA transfection. In order to determine the role of PU.1 in the expression of the Ccl22 gene, we introduced Spi1 siRNA into JAWSII and RAW264.7 cells. The introduction of Spi1 siRNA markedly decreased mRNA levels of Spi1 and Ccl22 in JAWSII (Fig. 3a) and RAW264.7 (Fig. 3b). Further, we confirmed the effect of Spi1 knockdown on Ccl22 expression in primary mouse DCs and macrophages generated from bone-marrow cells by cultivation in the presence of GM-CSF and M-CSF, respectively. Under the PU.1-knocked-down condition (both of mRNA and protein levels), the Ccl22 mRNA level was significantly decreased in BMDCs (Fig. 3c) and BMDMs (Fig. 3d). Furthermore, ELISA showed that the amount of CCL22 protein produced from BMDCs (Fig. 3e) and BMDMs (Fig. 3f) was markedly reduced by PU.1 knockdown. Although CCL22 production is enhanced during polarization to alternative activation macrophages (AAM), mRNA levels of other AAM makers, Fizz-1 and Ym-1 were not decreased but rather increased by Spi1 knockdown (Supplemental Fig. 2), suggesting that reduction of Ccl22 expression in Spi1 knockdown cells is not simply due to a reduction of these cells from a less of an AAM phenotype. To evaluate the effect of Spi1 knockdown-mediated suppression of CCL22 production on the capacity to cause migration of Th2 cells, we performed a migration assay. We observed that the number of Th2 cells migrated to Spi1-knocked-down DCs was moderately lower than that to control DCs (Supplemental Fig. 3). We introduced other Spi1 siRNAs into BMDCs to exclude the possibility of an off-target effect of Spi1 siRNA and found that these two siRNAs also significantly suppressed the Ccl22 mRNA level in parallel with the Spi1 knockdown level (Supplemental Fig. 4). Taken together, these results suggest that PU.1 is involved in the gene expression of mouse Ccl22 in DCs and macrophages.
Binding of PU.1 to the mouse Ccl22 promoter in DCs and macrophages. To investigate whether PU.1 binds to the Ccl22 gene in CCL22-expressing cells, we performed ChIP assays using BMDCs (Fig. 4a) and BMDMs (Fig. 4b). A significant amount of PU.1 binding to −17/+52 was detected in DCs and macrophages, whereas PU.1 did not bind to further upstream regions, −1780/−1714 and −1080/−1008 (Fig. 4a,b). These results demonstrate the specific binding of PU.1 to the proximal region of the transcription start site of the mouse Ccl22 gene. Furthermore, we carried out ChIP assays using DCs and macrophages freshly isolated from mice. As expected, significant and specific binding of PU.1 to −17/+52 of the Ccl22 promoter was detected in splenic DCs (Fig. 4c)  and peritoneal macrophages (Fig. 4d). These results indicate that PU.1 trasactivates the Ccl22 gene by binding to the proximal region of the promoter in DCs and macrophages in vivo.

Involvement of IRFs in the gene expression of Ccl22.
In addition to the role as a monomeric transcription factor, PU.1 also regulates the target genes forming a heterodimer with IRF4 or IRF8. In order to investigate the involvement of IRFs in the transcriptional regulation of Ccl22, we introduced siRNA against either Irf4 or Irf8 into BMDCs. Quantitative PCR showed that Irf4 knockdown significantly decreased the mRNA level of Ccl22 under the condition, in which levels of Irf4 mRNA and IRF4 protein were reduced (Fig. 5a). In contrast, Irf8 siRNA did not affect the Ccl22 mRNA level even though levels of Irf8 mRNA and IRF8 protein were decreased (Fig. 5b). To examine whether IRF4 co-localizes to the minimum promoter region of the Ccl22 gene with PU.1, we performed a ChIP assay using an anti-IRF4 Ab and quantified the amount of the immunoprecipitated chromosomal DNA by amplifying the region −17/+52. However, we did not detect significant binding of IRF4 around the identified PU.1 binding sites (Fig. 5c). We evaluated the effect of Irf4 siRNA on the expression and/or binding of PU.1 to −17/+52. As shown in Fig. 5d, levels of mRNA and protein of PU.1 were not affected by Irf4 siRNA. The amount of PU.1 binding to the region −17/+52 was not significantly reduced by Irf4 siRNA (Fig. 5e). These observations suggest that the involvement of IRF4 in the gene expression of Ccl22 is not due to the formation of a transcription complex with PU.1 around the transcriptional start site. IRF4 may transactivate the Ccl22 gene via another region on this gene or may indirectly regulate Ccl22 gene expression through transactivation of another transcription factor(s).

Effect of TLR-mediated stimulation on the recruitment of PU.1 to the gene. DCs and mac-
rophages dramatically alter their gene expression in response to stimulation signaling such as TLR ligands. As shown in Fig. 6a, the mRNA level of Ccl22 was markedly increased in response to LPS stimulation, in contrast to a slight increase in the Ccl17 mRNA level. Thus, we performed a ChIP assay to investigate whether the LPS-induced upregulation of Ccl22 transcription reflects the PU.1 binding ability to the Ccl22 promoter in DCs. As shown in Fig. 6b, the PU.1 binding level at the region −17/+52 in mature BMDCs was approximately 1.8-fold of that of immature BMDCs. We also confirmed that the LPS-induced increase of the Ccl22 mRNA level was significantly attenuated by siRNA-mediated Spi1 knockdown (Fig. 6c). These results demonstrate that PU.1 recruitment to the Ccl22 promoter is augmented by LPS-dependent maturation, thereby increasing the transcription of Ccl22.

Involvement of PU.1 in expression of the human CCL22 gene.
To investigate whether the role of PU.1 in Ccl22 expression in mouse monocytes is translatable to human biology, we evaluated the effect of SPI1 siRNA on CCL22 expression level in human monocyte cells, THP-1. As shown in Fig. 7a, SPI1 siRNA effectively suppressed SPI1 mRNA level and subsequently reduced CCL22 mRNA level. When nucleotide sequence of the human CCL22 gene was compared with that of mouse, we found that the elements similar to Site1 and Site2 exist on the human CCL22 promoter (Fig. 7b). By ChIP assay, we confirmed that PU.1 bound to this region in human monocyte cells (Fig. 7c). These results suggest that PU.1 is a common transactivator for the CCL22 (Ccl22) gene in human and mouse.

Discussion
The Ets family transcription factor PU.1 is essential for myeloid cell development. Several studies have demonstrated that PU.1 plays critical roles in DC and macrophage functions 13,14,16,20 . In the current study, we provided evidence that PU.1 is involved in Ccl22 expression in DCs and macrophages. Using a reporter assay, we demonstrated that transcriptional regulatory elements of the Ccl22 gene located within −27/+108 and two putative PU.1 binding elements were critical for transcriptional activity. Ccl22 expression was markedly decreased by Spi1 knockdown in all investigated cell types. ChIP assays showed that PU.1 bound to the proximal region of the Ccl22 promoter. From these results, we demonstrate that PU.1 is one of the most important transcription factors in the gene expression of Ccl22. Although transcriptional activity was significantly suppressed by mutation of the two PU.1 binding motifs in the luciferase assay, the activity was still higher  than that of those without the promoter. This result indicates that another transcription factor(s) is involved in the gene expression of Ccl22 by binding within the −27/+108 region. Indeed, CCL22 is also expressed in non-hematopoietic cells, such as keratinocytes, which do not express PU.1. Previous studies have demonstrated that NF-κB and STAT1 are involved in the IFN-γ/TNF-α-dependent upregulation of CCL22 in human keratinocytes 21,22 . Considering that our reporter assay was carried out in a steady state, these molecules were not likely to enhance the transcriptional activity via binding to the −27/+108 region. It will be intriguing to further investigating which transcription factors regulate Ccl22 gene expression. Even though ChIP assays were carried out using JAWSII and BMDCs without inducing maturation, we detected significant amount of PU.1 binding to the Ccl22 promoter. These results suggest that PU.1 plays a role as an activator of basal transcription of the Ccl22 gene in DCs, which produce a large amount of CCL22 even in the absence of inflammatory signaling. We also exhibited that PU.1 binding levels at the identified region were increased in response to LPS stimulation and subsequent upregulation of Ccl22 was significantly attenuated by Spi1 knockdown. These results suggest that PU.1 also plays an important role in the transactivation of Ccl22 upon DC maturation.
Here we used BMDMs and peritoneal macrophages as macrophage models. Macrophages are classified into two groups, M1 macrophages and M2 macrophages, depending on their expression of surface makers, cytokines, and chemokines. Ccl22 is one of the M2 macrophage-markers, and BMDMs generated with M-CSF are thought to exhibit an M2-like phenotype 23 . Indeed, we demonstrated that PU.1 is involved in the transcriptional regulation of Ccl22 by binding to the promoter in BMDMs. Recent studies have demonstrated that characteristic transcription factors like Spi-C and GATA6 are essential for differentiation of splenic and peritoneal macrophages, respectively 24,25 , suggesting that tissue-resident macrophages are functionally distinct. Although we showed that PU.1 marginally bound to the Ccl22 promoter in peritoneal macrophages, it is possible that PU.1 binding level is altered depending on where macrophages reside. Further analyses are required to elucidate this.
Previously, Chang et al. demonstrated that PU.1 promotes the development of Th9 26 and subpopulation of Th2 27 that express CCL22. PU.1 has also been described in promoting alternative activation of macrophages that produce CCL22 28 . In the current study, it was revealed that the CCL22 (Ccl22) gene, which has been reported to be under the control of PU.1, is directly transactivated by PU.1 via the cis-elements. Although Th cells were not examined in our study, the CCL22 gene may be a direct target of PU.1 in Th cells.
Previous studies have demonstrated that serum levels of CCL22 and CCL17 are significantly elevated and correlated with disease severity in patients with Atopic dermatitis 2,29-31 . Moreover, administration of CCR4 blocking antibody to the airway inflammation model abolished asthmatic symptoms 32 . According to these studies, targeting the interactions between CCL17/CCL22 and CCR4 may be a useful approach for controlling allergic diseases. We previously reported that injection of Spi1 siRNA significantly suppressed contact hypersensitivity of mice 13 . Considering that Th2-related genes such as Tnsf4 and Ccl22 are also transactivated by PU.1 in DCs, Spi1 knockdown may be a favorable strategy for allergic diseases.
Mouse DC line JAWSII, mouse macrophage line RAW264.7, and human monocyte cell line THP-1 were maintained as previously described 13,16,35 . All animal experiments were performed in accordance with the approved guidelines of the Institutional Review Board of Tokyo University of Science, Tokyo, Japan. The Animal Care and Use Committees of Tokyo University of Science specifically approved this study.
Isolation of splenic DCs and peritoneal macrophages. CD11c microbeads and an auto MACS Pro separator (Miltenyi Biotech, Tubingen, Germany) were used to isolate DCs from mouse spleen. Peritoneal macrophages were obtained as adhesive cells by incubating mouse peritoneal cells with 10% FCS-DMEM for 1 hour.