The powerful potential of amino acid menthyl esters for anti-inflammatory and anti-obesity therapies.

Our newly developed menthyl esters of valine and isoleucine exhibit anti-inflammatory properties beyond those of the well-known menthol in macrophages stimulated by lipopolysaccharide (LPS) and in a mouse model of colitis induced by sodium dextran sulfate. Unlike menthol, which acts primarily through the cold-sensitive TRPM8 channel, these menthyl esters displayed unique mechanisms that operate independently of this receptor. They readily penetrated target cells and efficiently suppressed LPS-stimulated tumour necrosis factor-alpha (Tnf) expression mediated by liver X receptor (LXR), a key nuclear receptor that regulates intracellular cholesterol and lipid balance. The menthyl esters showed affinity for LXR and enhanced the transcriptional activity through their non-competitive and potentially synergistic agonistic effect. This effect can be attributed to the crucial involvement of SCD1, an enzyme regulated by LXR, which is central to lipid metabolism and plays a key role in the anti-inflammatory response. In addition, we discovered that the menthyl esters showed remarkable efficacy in suppressing adipogenesis in 3T3-L1 adipocytes at the mitotic clonal expansion stage in an LXR-independent manner as well as in mice subjected to diet-induced obesity. These multiple capabilities of our compounds establish them as formidable allies in the fight against inflammation and obesity, paving the way for a range of potential therapeutic applications.

Fibroblast growth factor 18 stimulates the proliferation of hepatic stellate cells, thereby inducing liver fibrosis.

Liver fibrosis results from chronic liver injury triggered by factors such as viral infection, excess alcohol intake, and lipid accumulation. However, the mechanisms underlying liver fibrosis are not fully understood. Here, we demonstrate that the expression of fibroblast growth factor 18 (Fgf18) is elevated in mouse livers following the induction of chronic liver fibrosis models. Deletion of Fgf18 in hepatocytes attenuates liver fibrosis; conversely, overexpression of Fgf18 promotes liver fibrosis. Single-cell RNA sequencing reveals that overexpression of Fgf18 in hepatocytes results in an increase in the number of Lrat+ hepatic stellate cells (HSCs), thereby inducing fibrosis. Mechanistically, FGF18 stimulates the proliferation of HSCs by inducing the expression of Ccnd1. Moreover, the expression of FGF18 is correlated with the expression of profibrotic genes, such as COL1A1 and ACTA2, in human liver biopsy samples. Thus, FGF18 promotes liver fibrosis and could serve as a therapeutic target to treat liver fibrosis.

A rose flavor compound activating the NRF2 pathway in dendritic cells ameliorates contact hypersensitivity in mice.

Dendritic cells (DCs), which are typical antigen-presenting cells, localize to various sites in the body, particularly the front line of infection as sentinels, and are involved in innate and adaptive immune responses. Although the functions of DCs, such as pathogen-induced cytokine production and antigen-specific T cell activation, are important for host defenses against infection and tumorigenesis, the hyper- and/or extended activation of DCs leads to inflammatory and autoimmune diseases. In the present study, ß-damascone, a major ingredient of rose fragrance, was selected from an aroma library as a candidate compound that suppresses antigen-induced immune responses. ß-Damascone inhibited the functions of DCs, including the antigen-dependent proliferation of T cells, DC-induced Th1 development, and the TLR ligand-induced production of inflammatory cytokines by DCs. The ß-damascone treatment also increased the protein level of the transcription factor NF-E2-related factor 2 (NRF2), which plays key roles in antioxidant responses, and the transcription of Hmox1 and Nqo1, target genes of NRF2, in DCs. Nrf2 -/ - DCs induced Th1-development and produced large amount of IL-12p40 even in the presence of ß-damascone, whereas these functions by Nrf2 +/- DCs were inhibited by ß-damascone under the same conditions. The intake of ß-damascone suppressed ear swelling in contact hypersensitivity (CHS) model mice, but not in CHS-induced Nrf2 -/ - mice. Collectively, the present results indicate the potential of the rose aroma compound ß-damascone, which suppresses DC-mediated immune responses by activating the NRF2 pathway in DCs, for the prevention and/or attenuation of immune-mediated diseases.

Proscillaridin A Sensitizes Human Colon Cancer Cells to TRAIL-Induced Cell Death.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces cancer cell death by binding to TRAIL receptors. Because of its selective cytotoxicity toward cancer cells, TRAIL therapeutics, such as recombinant TRAIL and agonistic antibodies targeting TRAIL receptors, have garnered attention as promising cancer treatment agents. However, many cancer cells acquire resistance to TRAIL-induced cell death. To overcome this issue, we searched for agents to sensitize cancer cells to TRAIL-induced cell death by screening a small-molecule chemical library consisting of diverse compounds. We identified a cardiac glycoside, proscillaridin A, as the most effective TRAIL sensitizer in colon cancer cells. Proscillaridin A synergistically enhanced TRAIL-induced cell death in TRAIL-sensitive and -resistant colon cancer cells. Additionally, proscillaridin A enhanced cell death in cells treated with TRAIL and TRAIL sensitizer, the second mitochondria-derived activator of caspase mimetic. Proscillaridin A upregulated TRAIL receptor expression, while downregulating the levels of the anti-cell death molecules, cellular FADD-like IL-1ß converting enzyme-like inhibitor protein and Mcl1, in a cell type-dependent manner. Furthermore, proscillaridin A enhanced TRAIL-induced cell death partly via O-glycosylation. Taken together, our findings suggest that proscillaridin A is a promising agent that enhances the anti-cancer efficacy of TRAIL therapeutics.

Delta Opioid Receptor Agonists Ameliorate Colonic Inflammation by Modulating Immune Responses.

The opioid receptors play important roles in the regulation of sense and emotions. Although it is recently revealed that opioid receptors are also expressed in various cells, but not restricted in the central nervous system, the effects of opioids on peripheral immune cells are largely unknown. In the current study, we evaluated the effect of opioids on immune system by using selective agonists for δ opioid receptor. Systemic administration of KNT-127 or intraperitoneal injection of YNT-2715 (a KNT-127-related compound that cannot pass through the blood-brain barrier) significantly alleviated the pathology of dextran sodium sulfate-induced colitis. In KNT-127-treated mice, the levels of an inflammatory cytokine IL-6 in the serum, and macrophages in the mesenteric lymph nodes (MLNs) were decreased in the progression stage, and those of regulatory T cells (Tregs) in the MLN were increased in the recovery stage. In vitro experiments revealed that KNT-127 inhibited the release of IL-6 and another inflammatory cytokine TNF-α from macrophages and accelerated the development of Tregs. Our study suggests that δ opioid agonists act directly on immune cells to improve the pathology of the colitis and can be candidates of immunomodulatory drugs.

IL-10 in Mast Cell-Mediated Immune Responses: Anti-Inflammatory and Proinflammatory Roles.

Mast cells (MCs) play critical roles in Th2 immune responses, including the defense against parasitic infections and the initiation of type I allergic reactions. In addition, MCs are involved in several immune-related responses, including those in bacterial infections, autoimmune diseases, inflammatory bowel diseases, cancers, allograft rejections, and lifestyle diseases. Whereas antigen-specific IgE is a well-known activator of MCs, which express FcεRI on the cell surface, other receptors for cytokines, growth factors, pathogen-associated molecular patterns, and damage-associated molecular patterns also function as triggers of MC stimulation, resulting in the release of chemical mediators, eicosanoids, and various cytokines. In this review, we focus on the role of interleukin (IL)-10, an anti-inflammatory cytokine, in MC-mediated immune responses, in which MCs play roles not only as initiators of the immune response but also as suppressors of excessive inflammation. IL-10 exhibits diverse effects on the proliferation, differentiation, survival, and activation of MCs in vivo and in vitro. Furthermore, IL-10 derived from MCs exerts beneficial and detrimental effects on the maintenance of tissue homeostasis and in several immune-related diseases including contact hypersensitivity, auto-immune diseases, and infections. This review introduces the effects of IL-10 on various events in MCs, and the roles of MCs in IL-10-related immune responses and as a source of IL-10.

PU.1 and IRF8 Modulate Activation of NLRP3 Inflammasome via Regulating Its Expression in Human Macrophages.

NLRP3 inflammasomes play crucial roles in the initiation of host defense by converting pro-Caspase-1 to mature Caspase-1, which in turn processes immature IL-1ß and IL-18 into their biologically active forms. Although NLRP3 expression is restricted to monocytic lineages such as monocytes, macrophages, and dendritic cells, the mechanisms determining the lineage-specific expression of NLRP3 remain largely unknown. In this study, we investigated the transcription factors involved in cell-type-specific transcription of NLRP3. We found that a distal, rather than a proximal, promoter of human NLRP3 was predominantly used in the human monocytic cell lines and macrophages. Reporter analysis showed that an Ets/IRF composite element (EICE) at -309/-300 and an Ets motif at +5/+8 were critical for transcriptional activity of the distal promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that two transcription factors, PU.1 and IRF8, both of which play essential roles in development and gene expression of the monocytic lineage, were bound to the EICE site, whereas PU.1 alone was bound to the Ets site. Knockdown of PU.1 and/or IRF8 mediated by small interfering RNA downregulated expression of NLRP3 and related molecules and markedly diminished the LPS-induced release of IL-1ß in THP-1, suggesting that activity of the NLRP3 inflammasome was suppressed by knockdown of PU.1 and IRF8. Taken together, these results indicate that PU.1 and IRF8 are involved in the monocytic lineage-specific expression of NLRP3 by binding to regulatory elements within its promoter and that PU.1 and IRF8 are potential targets for regulating the activity of the NLRP3 inflammasome.

Interleukin-11-expressing fibroblasts have a unique gene signature correlated with poor prognosis of colorectal cancer.

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.

Pterostilbene reduces colonic inflammation by suppressing dendritic cell activation and promoting regulatory T cell development.

Dendritic cells (DCs) and T cells play important roles in immune regulation, and modulating their function is an approach for developing preventive or therapeutic strategies against immune disorders. Herein, the effect of pterostilbene (PSB) (3',5'-dimethoxy-resveratrol)-a resveratrol-related polyphenol found in blueberries-on immune regulation was evaluated. Using an in vitro co-culture system, PSB was found to exert the strongest inhibitory effect among all tested resveratrol derivatives on DC-mediated T cell proliferation; moreover, PSB treatment decreased the Th1 and Th17 populations and increased the regulatory T cell (Treg) population. Upon co-stimulation with anti-CD3 and anti-CD28 antibodies, PSB inhibited CD4+ T cell proliferation and differentiation into Th1 cells. Additionally, PSB acted on DCs to suppress the lipopolysaccharide-induced transactivation of genes encoding antigen presentation-related molecules and inflammatory cytokines by attenuating the DNA-binding ability of the transcription factor PU.1. Furthermore, PSB promoted DC-mediated Foxp3+ Treg differentiation, and PU.1 knockdown increased DC-induced Treg activity. Oral administration of PSB alleviated the symptoms of dextran sulfate sodium-induced colitis and decreased tumor necrosis factor-α expression in mice. Thus, PSB treatment ameliorates colonic inflammation.

The anti-inflammatory effect of the gut lactic acid bacteria-generated metabolite 10-oxo-cis-6,trans-11-octadecadienoic acid on monocytes.

We evaluated the effect of gut bacterial metabolites of polyunsaturated fatty acids on inflammation and found that 10-oxo-cis-6,trans-11-octadecadienoic acid (γKetoC) strikingly suppressed LPS-induced IL-6 release from bone marrow-derived macrophages (BMMs), which was accompanied by reduced mRNA expression of Il6, TNF, and Il1b. γKetoC decreased the cAMP concentration in BMMs, suggesting that γKetoC stimulated G protein-coupled receptors. A Gq agonist significantly suppressed LPS-induced IL-6 expression in BMMs, whereas a Gi inhibitor partially abrogated γKetoC-mediated IL-6 suppression. Cytosolic Ca2+ was markedly increased by γKetoC, which was partly but not fully abrogated by an ion channel inhibitor. Taken together, these data suggest that γKetoC suppresses inflammatory cytokine expression in macrophages primarily through Gq and partially through Gi. γKetoC suppressed osteoclast development and IL-6 expression in synovial fibroblasts from rheumatoid arthritis (RA) patients, suggesting the beneficial effect of γKetoC on the prevention or treatment of RA.

Dual Roles of PU.1 in the Expression of PD-L2: Direct Transactivation with IRF4 and Indirect Epigenetic Regulation.

PD-L2, which has been identified as a PD-1 ligand, is specifically expressed in dendritic cells (DCs) and macrophages. The transcription factors that determine the cell type-specific expression of PD-L2 are largely unknown, although PD-1 and its ligands, which have been shown to play important roles in T cell suppression, have been vigorously analyzed in the field of cancer immunology. To reveal the mechanism by which Pdcd1lg2 gene expression is regulated, we focused on DCs, which play key roles in innate and acquired immunity. The knockdown of the hematopoietic cell-specific transcription factors PU.1 and IRF4 decreased PD-L2 expression in GM-CSF-induced mouse bone marrow-derived DCs. Chromatin immunoprecipitation assays, luciferase assays, and electrophoretic mobility shift assays demonstrated that PU.1 and IRF4 bound directly to the Pdcd1lg2 gene via an Ets-IRF composite element sequence and coordinately transactivated the Pdcd1lg2 gene. Furthermore, PU.1 knockdown reduced the histone acetylation of the Pdcd1lg2 gene. The knockdown of the typical histone acetyltransferase p300, which has been reported to interact with PU.1, decreased the expression and H3K27 acetylation of the Pdcd1lg2 gene. GM-CSF stimulation upregulated the Pdcd1lg2 gene expression, which was accompanied by an increase in PU.1 binding and histone acetylation in Flt3L-generated mouse bone marrow-derived DCs. The involvement of PU.1, IRF4, and p300 were also observed in mouse splenic DCs. Overall, these results indicate that PU.1 positively regulates Pdcd1lg2 gene expression as a transactivator and an epigenetic regulator in DCs.

PU.1 regulates Ccr7 gene expression by binding to its promoter in naïve CD4+ T cells.

C-C chemokine receptor type 7 (CCR7) is expressed on naïve T cells, B cells, and activated dendritic cells (DCs). We previously demonstrated that the transcription factor PU.1/Spi1 positively regulates the expression of CCR7 in DCs. In the present study, we investigated the role of PU.1 in CCR7 expression in T cells. To confirm whether PU.1 is involved in the expression of CCR7, we conducted a ChIP assay in various T cells purified from splenocytes and thymocytes and found that PU.1 binds to the Ccr7 promoter-proximal region in spleen naïve CD4+ T cells, but not in thymocytes. Small interfering RNA-mediated PU.1 knockdown resulted in decreased CCR7 expression in spleen naïve CD4+ T cells. Compared to naïve CD4+ T cells, Spi1 and Ccr7 mRNA levels decreased in Th1 and Th2 cells, in which PU.1 did not bind to the Ccr7 promoter, suggesting that CCR7 expression decreases due to the dissociation of PU.1 from the Ccr7 promoter during the development of effector T cells from naïve T cells. Collectively, we concluded that CCR7 expression level correlates with the binding level of PU.1 to the Ccr7 promoter and PU.1 acts as a transcriptional activator of the Ccr7 gene in naïve CD4+ T cells.

Cooperative Regulation of the Mucosal Mast Cell-Specific Protease Genes Mcpt1 and Mcpt2 by GATA and Smad Transcription Factors.

Mouse mast cell proteases (mMCP)-1 and -2 are specifically expressed in mucosal mast cells (MCs). However, the transcriptional regulation mechanism of the Mcpt1 and Mcpt2 genes induced in mucosal MCs is largely unknown. In the current study, we found that TGF-ß stimulation drastically induced upregulation of Mcpt1 and Mcpt2 mRNA in mouse bone marrow-derived MCs (BMMCs). TGF-ß-induced expression of Mcpt1 and Mcpt2 was markedly suppressed by transfection with small interfering RNA targeting Smad2 or Smad4 and moderately reduced by Smad3 small interfering RNA. We next examined the roles of the hematopoietic cell-specific transcription factors GATA1 and GATA2 in the expression of Mcpt1 and Mcpt2 and demonstrated that knockdown of GATA1 and GATA2 reduced the mRNA levels of Mcpt1 and Mcpt2 in BMMCs. The recruitment of GATA2 and acetylation of histone H4 of the highly conserved GATA-Smad motifs, which were localized in the distal regions of the Mcpt1 and Mcpt2 genes, were markedly increased by TGF-ß stimulation, whereas the level of GATA2 binding to the proximal GATA motif was not affected by TGF-ß. A reporter assay showed that TGF-ß stimulation upregulated GATA2-mediated transactivation activity in a GATA-Smad motif-dependent manner. We also observed that GATA2 and Smad4 interacted in TGF-ß-stimulated BMMCs via immunoprecipitation and Western blotting analysis. Taken together, these results demonstrate that TGF-ß induced mMCP-1 and -2 expression by accelerating the recruitment of GATA2 to the proximal regions of the Mcpt1 and Mcpt2 genes in mucosal MCs.

Suppressive role of PPARγ in the IgE-dependent activation of mast cells.

Mast cells (MCs) play a central role in IgE-dependent immune responses. PPARγ is a nuclear receptor that is essential for adipocyte differentiation and insulin sensitivity. Although PPARγ is expressed in activated MCs, the effect of PPARγ suppression in IgE-mediated activation of MCs is largely unknown. In the current study, we evaluated the effect of PPARγ knockdown on the function of IgE plus antigen (Ag)-stimulated MCs using siRNA-transfected bone marrow-derived MCs (BMMCs). We found that the mRNA expression level of cytokines in IgE/Ag-stimulated BMMCs was significantly increased in PPARγ knockdown BMMCs, and IgE/Ag-mediated degranulation and the protein production level of TNF-α was moderately increased by PPARγ knockdown, whereas the cell surface expression level of FcεRI was not affected by PPARγ knockdown. Oral administration of pioglitazone (PPARγ agonist) significantly suppressed body temperature change of mice in passive systemic anaphylaxis, supporting the inhibitory functions of PPARγ in IgE/Ag-dependent activation of MCs in vivo. IgE-mediated up-regulation of mRNA levels of Ptgs2 (encoding COX-2) was drastically enhanced in PPARγ knockdown BMMCs. Although several prostaglandin (PG) derivatives are known to be ligands for PPARγ, treatment with a COX inhibitor, acetyl salicylic acid, up-regulated the IgE-mediated increase of Il13, Tnf and Ptgs2 mRNA levels in a synergistic manner with PPARγ siRNA. Knockdown of COX-1 and/or COX-2 by siRNA showed that suppression of IgE/Ag-mediated activation was mainly dependent on COX-1. Taken together, these results indicate that PPARγ suppresses IgE/Ag-induced transactivation of cytokine genes and the Ptgs2 gene in MCs in a manner distinguishable from that of PGs.

PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

C-C chemokine receptor type 7 (CCR7) is essential for migration of dendritic cells (DCs) to draining lymph nodes. PU.1/Spi1 is a transcription factor playing a critical role in the gene regulation of DCs. PU.1 knockdown decreased the expression of CCR7 in bone marrow-derived DCs and subsequently attenuated migration in vitro and in vivo. Reporter assays, EMSA, and chromatin immunoprecipitation assays revealed that PU.1 binds to the most proximal Ets motif of the Ccr7 promoter, which is involved in transcriptional activation. The CCR7 expression level, which was higher in the programmed cell death 1 ligand 2 (PD-L2)+ population than in the PD-L2- population and was markedly suppressed by TGF-ß treatment, coincided with the binding level of PU.1 to the Ccr7 promoter. The PU.1 binding level in CCR7high mesenteric lymph nodes DCs was higher than in other DC subtypes. The involvement of PU.1 in the expression of the CCR7 gene was also observed in human DCs. We conclude that PU.1 plays a pivotal role in DC migration by transactivating the CCR7 gene via the Ets motif in the promoter in both humans and mice.-Yashiro, T., Takeuchi, H., Nakamura, S., Tanabe, A., Hara, M., Uchida, K., Okumura, K., Kasakura, K., Nishiyama, C. PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

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.

Blockade of TNF receptor superfamily 1 (TNFR1)-dependent and TNFR1-independent cell death is crucial for normal epidermal differentiation.

BACKGROUND: A delicate balance between cell death and keratinocyte proliferation is crucial for normal skin development. Previous studies have reported that cellular FLICE (FADD-like ICE)-inhibitory protein plays a crucial role in prevention of keratinocytes from TNF-α-dependent apoptosis and blocking of dermatitis. However, a role for cellular FLICE-inhibitory protein in TNF-α-independent cell death remains unclear. OBJECTIVE: We investigated contribution of TNF-α-dependent and TNF-α-independent signals to the development of dermatitis in epidermis-specific Cflar-deficient (CflarE-KO) mice. METHODS: We examined the histology and expression of epidermal differentiation markers and inflammatory cytokines in the skin of CflarE-KO;Tnfrsf1a+/- and CflarE-KO;Tnfrsf1a-/- mice. Mice were treated with neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand to block TNF-α-independent cell death of CflarE-KO;Tnfrsf1a-/- mice. RESULTS: CflarE-KO;Tnfrsf1a-/- mice were born but experienced severe dermatitis and succumbed soon after birth. CflarE-KO;Tnfrsf1a+/- mice exhibited embryonic lethality caused by massive keratinocyte apoptosis. Although keratinocytes from CflarE-KO;Tnfrsf1a-/- mice still died of apoptosis, neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand substantially prolonged survival of CflarE-KO;Tnfrsf1a-/- mice. Expression of inflammatory cytokines, such as Il6 and Il17a was increased; conversely, expression of epidermal differentiation markers was severely downregulated in the skin of CflarE-KO;Tnfrsf1a-/- mice. Treatment of primary keratinocytes with IL-6 and, to a lesser extent, IL-17A suppressed expression of epidermal differentiation markers. CONCLUSION: TNF receptor superfamily 1 (TNFR1)-dependent or TNFR1-independent apoptosis of keratinocytes promotes inflammatory cytokine production, which subsequently blocks epidermal differentiation. Thus blockade of both TNFR1-dependent and TNFR1-independent cell death might be an alternative strategy to treat skin diseases when treatment with anti-TNF-α antibody alone is not sufficient.

Transcription factor Tlx1 marks a subset of lymphoid tissue organizer-like mesenchymal progenitor cells in the neonatal spleen.

The spleen is comprised of spatially distinct compartments whose functions, such as immune responses and removal of aged red blood cells, are tightly controlled by the non-hematopoietic stromal cells that provide regionally-restricted signals to properly activate hematopoietic cells residing in each area. However, information regarding the ontogeny and relationships of the different stromal cell types remains limited. Here we have used in vivo lineage tracing analysis and in vitro mesenchymal stromal cell assays and found that Tlx1, a transcription factor essential for embryonic spleen organogenesis, marks neonatal stromal cells that are selectively localized in the spleen and retain mesenchymal progenitor potential to differentiate into mature follicular dendritic cells, fibroblastic reticular cells and marginal reticular cells. Furthermore, by establishing a novel three-dimensional cell culture system that enables maintenance of Tlx1-expressing cells in vitro, we discovered that signals from the lymphotoxin ß receptor and TNF receptor promote differentiation of these cells to express MAdCAM-1, CCL19 and CXCL13, representative functional molecules expressed by different subsets of mature stromal cells in the spleen. Taken together, these findings indicate that mesenchymal progenitor cells expressing Tlx1 are a subset of lymphoid tissue organizer-like cells selectively found in the neonatal spleen.

The Transcription Factors PU.1 and IRF4 Determine Dendritic Cell-Specific Expression of RALDH2.

RALDH2 expressed in dendritic cells (DCs) plays a critical role in the development of regulatory T cells in mesenteric lymph nodes. Despite the importance of RALDH2 in intestinal immunity, little is known about the mechanism of DC-specific expression of RALDH2. In the current study, we focused on the hematopoietic cell-specific transcription factors PU.1 and IRF4 as the determinants of Aldh1a2 gene expression. The mRNA level of Aldh1a2, and subsequently the enzyme activity, were decreased by knockdown of PU.1 and IRF4 in bone marrow-derived DCs (BMDCs) of BALB/c mice. Chromatin immunoprecipitation assays showed that PU.1 and IRF4 bound to the Aldh1a2 gene ∼2 kb upstream from the transcription start site in BMDCs. A reporter assay and an EMSA revealed that the Aldh1a2 promoter was synergistically transactivated by a heterodimer composed with PU.1 and IRF4 via the EICE motif at -1961/-1952 of the gene. The effect of small interfering RNAs for Spi1 and Irf4 and specific binding of PU.1 and IRF4 on the Aldh1a2 gene were also observed in DCs freshly isolated from spleen and mesenteric lymph nodes, respectively. GM-CSF stimulation upregulated the Aldh1a2 transcription in Flt3 ligand-generated BMDCs, in which the IRF4 expression and the PU.1 recruitment to the Aldh1a2 promoter were enhanced. We conclude that PU.1 and IRF4 are transactivators of the Aldh1a2 gene in vitro and ex vivo.