The aging mouse lens transcriptome.

Age is a major risk factor for cataract (ARC). However, the influence of aging on the lens transcriptome is under studied. Lens epithelial (LEC) and fiber cells (LFC) were isolated from young (3 month old) and aged (24 month old) C57BL/6J mice, and the transcriptome elucidated via RNAseq. EdgeR estimated differential gene expression in pairwise contrasts, and Advaita's Ipathway guide and custom R scripts were used to evaluate the potential biological significance of differentially expressed genes (DEGs). This analysis revealed age-dependent decreases in lens differentiation marker expression in both LECs and LFCs, with gamma crystallin transcripts downregulating nearly 50 fold in aged LFCs. The expression of the transcription factors Hsf4 and Maf, which are known to activate lens fiber cell preferred genes, are downregulated, while FoxE3, which represses gamma crystallin expression, is upregulated in aged fibers. Aged LECs upregulate genes controlling the immune response, complement pathways, and cellular stress responses, including glutathione peroxidase 3 (Gpx3). Aged LFCs exhibit broad changes in the expression of genes regulating cell communication, and upregulate genes involved in antigen processing/presentation and cholesterol metabolism, while changes in the expression of mitochondrial respiratory chain genes are consistent with mitochondrial stress, including upregulation of NDufa4l2, which encodes an alternate electron transport chain protein. However, age did not profoundly affect the response of LECs to injury as both young and aged LECs upregulate inflammatory gene signatures at 24 h post injury to similar extents. These RNAseq profiles provide a rich data set that can be mined to understand the genetic regulation of lens aging and how this impinges on the pathophysiology of age related cataract.

Circulating microRNA Expression Profiling Identifies miR-125a-5p Promoting T Helper 1 Cells Response in the Pathogenesis of Hashimoto's Thyroiditis.

MicroRNAs (miRNAs) have emerged as key regulators of cellular processes by suppressing target mRNAs at the posttranscriptional level. However, little is known regarding the expression of miRNAs in peripheral blood mononuclear cells (PBMCs) from Hashimoto's thyroiditis (HT) patients. Therefore, 38 HT patients and 36 healthy volunteers were enrolled in this study to identify HT-mediated changes in miRNA expression. Over 1,000 dysregulated miRNAs and their biological functions in the HT patients were identified. Among them, miR-125a-5p expression was upregulated and inversely correlated with low levels of MAF, a transcription factor that inhibits Th1 cells activity and the production of IFN-γ. Luciferase assay results demonstrated that MAF is a direct target gene of miR-125a-5p. Moreover, the proportion of circulating Th1 cells and the transcript levels of IFN-γ were increased in the HT patients. MiR-125a-5p expression positively correlated with the proportion of circulating Th1 cells and the serum concentrations of anti-thyroperoxidase antibodies in the HT patients. Interestingly, knockdown of miR-125a-5p in CD4+ T cells resulted in an elevated level of MAF but decreased the proportion of Th1 cells and the transcript level of IFN-γ in vitro. Furthermore, upregulated miR-125a-5p and IFN-γ transcript levels and downregulated MAF expression were detected in thyroid tissues from HT patients. Receiver operating characteristic (ROC) curves suggested that miR-125a-5p has a crucial role in the HT. Our results demonstrate that the elevated levels of miR-125a-5p contribute to the Th1 cells response in the HT patients and may be involved in the pathogenesis of HT.

Maf expression in human macrophages and lymph node sinus macrophages in patients with esophageal cancer.

The large Maf transcription factors are expressed in immune cells including macrophages and lymphocytes. To investigate the distribution of Maf expression in human organs, immunostaining for Maf was performed using sections of several human organs. High Maf expression was seen in the nucleus of macrophages in the gastrointestinal tract and lymph node sinus macrophages (LySMs). Then, we assessed whether Maf expression in LySMs was correlated with CD169 expression and the clinical prognosis in patients with esophageal cancer. Maf expression was associated with CD169 expression, but Maf expression in LySMs was not associated with the clinical course in patients with esophageal cancer. We determined which cytokines stimulate Maf expression using cultured macrophages. Immunocytochemistry showed that Maf expression was significantly elevated by interferon-γ. These results are the first report of Maf expression in human samples. Maf expression may be a marker for the macrophage population in humans.

ß1-integrin controls cell fate specification in early lens development.

Integrins are heterodimeric cell surface molecules that mediate cell-extracellular matrix (ECM) adhesion, ECM assembly, and regulation of both ECM and growth factor induced signaling. However, the developmental context of these diverse functions is not clear. Loss of ß1-integrin from the lens vesicle (mouse E10.5) results in abnormal exit of anterior lens epithelial cells (LECs) from the cell cycle and their aberrant elongation toward the presumptive cornea by E12.5. These cells lose expression of LEC markers and initiate expression of the Maf (also known as c-Maf) and Prox1 transcription factors as well as other lens fiber cell markers. ß1-integrin null LECs also upregulate the ERK, AKT and Smad1/5/8 phosphorylation indicative of BMP and FGF signaling. By E14.5, ß1-integrin null lenses have undergone a complete conversion of all lens epithelial cells into fiber cells. These data suggest that shortly after lens vesicle closure, ß1-integrin blocks inappropriate differentiation of the lens epithelium into fibers, potentially by inhibiting BMP and/or FGF receptor activation. Thus, ß1-integrin has an important role in fine-tuning the response of the early lens to the gradient of growth factors that regulate lens fiber cell differentiation.

Regulation of c-Maf and αA-Crystallin in Ocular Lens by Fibroblast Growth Factor Signaling.

Fibroblast growth factor (FGF) signaling regulates a multitude of cellular processes, including cell proliferation, survival, migration, and differentiation. In the vertebrate lens, FGF signaling regulates fiber cell differentiation characterized by high expression of crystallin proteins. However, a direct link between FGF signaling and crystallin gene transcriptional machinery remains to be established. Previously, we have shown that the bZIP proto-oncogene c-Maf regulates expression of αA-crystallin (Cryaa) through binding to its promoter and distal enhancer, DCR1, both activated by FGF2 in cell culture. Herein, we identified and characterized a novel FGF2-responsive region in the c-Maf promoter (-272/-70, FRE). Both c-Maf and Cryaa regulatory regions contain arrays of AP-1 and Ets-binding sites. Chromatin immunoprecipitation (ChIP) assays established binding of c-Jun (an AP-1 factor) and Etv5/ERM (an Ets factor) to these regions in lens chromatin. Analysis of temporal and spatial expression of c-Jun, phospho-c-Jun, and Etv5/ERM in wild type and ERK1/2 deficient lenses supports their roles as nuclear effectors of FGF signaling in mouse embryonic lens. Collectively, these studies show that FGF signaling up-regulates expression of αA-crystallin both directly and indirectly via up-regulation of c-Maf. These molecular mechanisms are applicable for other crystallins and genes highly expressed in terminally differentiated lens fibers.

Cell-Intrinsic gp130 Signaling on CD4+ T Cells Shapes Long-Lasting Antiviral Immunity.

The IL-6 cytokine family utilizes the common signal transduction molecule gp130, which can mediate a diverse range of outcomes. To clarify the role of gp130 signaling in vivo during acute viral infection, we infected Cd4-cre Il6st(fl/fl) mice, in which gp130 is conditionally ablated in T cells, with acute lymphocytic choriomeningitis virus. We found that by day 12, but not at day 8, after infection the number of virus-specific CD4(+) T cells was reduced in the absence of gp130, and this was sustained for up to 2 mo postinfection. Additionally, gp130-deficient T follicular helper cells had lower expression of Maf, IL-21, and ICOS, and this was accompanied by a reduction in the proportion of germinal center B cells and plasmablasts. Remarkably, at 2 mo postinfection the proportion of IgG2a/c(+) memory B cells and the systemic levels of lymphocytic choriomeningitis virus-specific IgG2 Abs were dramatically decreased, whereas there was a corresponding increase in IgG1(+) memory B cells and virus-specific IgG1 Abs. In the same animals gp130-deficient virus-specific CD8(+) T cells showed a reduced proportion of memory cells, which expressed lower levels of Tcf7, and displayed diminished recall responses on secondary infection. Mixed bone marrow chimeras revealed that the aforementioned gp130 effects on CD4(+) T cells were cell intrinsic. Overall, our data show that gp130 signaling in T cells influences the quantity and quality of long-lasting CD4(+) T cell responses as well as CD8(+) T cell- and Ab-mediated immunity after acute viral infection.

Glycogen synthase kinase-3 controls IL-10 expression in CD4(+) effector T-cell subsets through epigenetic modification of the IL-10 promoter.

The serine/threonine kinase glycogen synthase kinase-3 (GSK3) plays an important role in balancing pro- and anti-inflammatory cytokines. We have examined the role of GSK3 in production of IL-10 by subsets of CD4(+) T helper cells. Treatment of naive murine CD4(+) T cells with GSK3 inhibitors did not affect their production of IL-10. However, treatment of Th1 and Th2 cells with GSK3 inhibitors dramatically increased production of IL-10. GSK3 inhibition also led to upregulation of IL-10 among Th1, Th2, and Th17 subsets isolated from human blood. The encephalitogenic potential of GSK3 inhibitor treated murine Th1 cells was significantly reduced in adoptive transfer experiments by an IL-10-dependent mechanism. Analysis of the murine IL-10 promoter in response to inhibition of GSK3 in Th1 cells showed modification to a transcriptionally active state indicated by changes in histone H3 acetylation and methylation. Additionally, GSK3 inhibition increased expression of the transcription factors c-Maf, Nfil3, and GATA3, correlating with the increase in IL-10. These findings are important in the context of autoimmune disease since they show that it is possible to reprogram disease-causing cells through GSK3 inhibition.

Interleukin-21-producing c-Maf-expressing CD4+ T cells induce effector CD8+ T cells and enhance autoimmune inflammation in scurfy mice.

OBJECTIVE: FoxP3 induces Treg cells and prevents autoimmune diseases. However, the precise mechanisms of FoxP3 in the prevention of autoimmune diseases remain unknown. We undertook this study to determine the regulatory roles of FoxP3 in autoimmune inflammation by using FoxP3-mutant sf mice. METHODS: We characterized interleukin-21 (IL-21)- producing cells in sf mice. We examined the underlying mechanisms of enhanced IL-21 production in sf mouse CD4+ T cells. We examined the roles of IL-21 and CD8+ T cells in autoimmune inflammation in sf mice using IL-21 receptor (IL-21R)-deficient sf mice. RESULTS: IL-21-producing c-Maf+CD4+ T cells, which were distinct from Th17 cells, were increased in sf mice. Increased c-Maf expression was involved in enhanced IL-21 production in sf mouse CD4+ T cells. Experiments using bone marrow chimeric mice showed that lack of cell-extrinsic suppression by FoxP3+ Treg cells, but not cell-intrinsic defects in FoxP3 in sf mouse CD4+ T cells, was mainly involved in the development of IL-21-producing c-Maf+CD4+ T cells in sf mice. IL-21R deficiency prolonged survival and reduced multiorgan autoimmune inflammation in sf mice. Moreover, IL-21R deficiency decreased short-lived effector CD8+ T cells in the lung in sf mice. Furthermore, depletion of CD8+ T cells inhibited lung inflammation in sf mice, suggesting that CD8+ T cells are critical for inducing lung inflammation in sf mice. CONCLUSION: Unique IL-21-producing c-Maf+ CD4+ T cells develop in the absence of FoxP3+ Treg cells, induce short-lived effector CD8+ T cells, and enhance multiorgan autoimmune inflammation in sf mice.

Activation of the hedgehog signaling pathway in the developing lens stimulates ectopic FoxE3 expression and disruption in fiber cell differentiation.

PURPOSE: The signaling pathways and transcriptional effectors responsible for directing mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens. METHODS: We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm. RESULTS: Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death. CONCLUSIONS: Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3.

IL-2 regulates expression of C-MAF in human CD4 T cells.

Blockade of IL-2R with humanized anti-CD25 Abs, such as daclizumab, inhibits Th2 responses in human T cells. Recent murine studies have shown that IL-2 also plays a significant role in regulating Th2 cell differentiation by activated STAT5. To explore the role of activated STAT5 in the Th2 differentiation of primary human T cells, we studied the mechanisms underlying IL-2 regulation of C-MAF expression. Chromatin immunoprecipitation studies revealed that IL-2 induced STAT5 binding to specific sites in the C-MAF promoter. These sites corresponded to regions enriched for markers of chromatin architectural features in both resting CD4 and differentiated Th2 cells. Unlike IL-6, IL-2 induced C-MAF expression in CD4 T cells with or without prior TCR stimulation. TCR-induced C-MAF expression was significantly inhibited by treatment with daclizumab or a JAK3 inhibitor, R333. Furthermore, IL-2 and IL-6 synergistically induced C-MAF expression in TCR-activated T cells, suggesting functional cooperation between these cytokines. Finally, both TCR-induced early IL4 mRNA expression and IL-4 cytokine expression in differentiated Th2 cells were significantly inhibited by IL-2R blockade. Thus, our findings demonstrate the importance of IL-2 in Th2 differentiation in human T cells and support the notion that IL-2R-directed therapies may have utility in the treatment of allergic disorders.

A novel transgenic mouse model of the human multiple myeloma chromosomal translocation t(14;16)(q32;q23).

Multiple myeloma (MM) is a currently incurable neoplasm of terminally differentiated B cells. The translocation and/or overexpression of c-MAF have been observed in human MM. Although c-MAF might function as an oncogene in human MM, there has been no report thus far describing the direct induction of MM by c-MAF overexpression in vivo. In this study, we have generated transgenic (TG) mice that express c-Maf specifically in the B-cell compartment. Aged c-Maf TG mice developed B-cell lymphomas with some clinical features that resembled those of MM, namely, plasma cell expansion and hyperglobulinemia. Quantitative RT-PCR analysis demonstrated that Ccnd2 and Itgb7, which are known target genes of c-Maf, were highly expressed in the lymphoma cells. This novel TG mouse model of the human MM t(14;16)(q32;q23) chromosomal translocation should serve to provide new insight into the role of c-MAF in tumorigenesis.

c-Maf-dependent growth of Mycobacterium tuberculosis in a CD14(hi) subpopulation of monocyte-derived macrophages.

Macrophages are a major component of the innate immune response, comprising the first line of defense against various intracellular pathogens, including Mycobacterium tuberculosis. In this report, we studied the factors that regulate growth of M. tuberculosis H37Rv in subpopulations of human monocyte-derived macrophages (MDMs). In healthy donors, M. tuberculosis H37Rv grew 5.6-fold more rapidly in CD14(hi) MDMs compared with that in CD14(lo)CD16(+) MDMs. Compared with CD14(lo)CD16(+) cells, M. tuberculosis H37Rv-stimulated CD14(hi) monocytes produced more IL-10 and had increased mRNA expression for c-Maf, a transcription factor that upregulates IL-10 gene expression. c-Maf small interfering RNA (siRNA) inhibited IL-10 production and growth of M. tuberculosis in CD14(hi) cells. Compared with CD14(lo)CD16(+) monocytes, M. tuberculosis H37Rv-stimulated CD14(hi) cells had increased expression of 22 genes whose promoters contained a c-Maf binding site, including hyaluronan synthase 1 (HAS1). c-Maf siRNA inhibited HAS1 expression in M. tuberculosis-stimulated CD14(hi) monocytes, and HAS1 siRNA inhibited growth of M. tuberculosis in CD14(hi) MDMs. M. tuberculosis H37Rv upregulated expression of HAS1 protein and its product, hyaluronan, in CD14(hi) MDMs. We conclude that M. tuberculosis grows more rapidly in CD14(hi) than in CD14(lo)CD16(+) MDMs because CD14(hi) cells have increased expression of c-Maf, which increases production of two key factors (hyaluronan and IL-10) that promote growth of M. tuberculosis.

Frequent downregulation or loss of CD79a expression in plasma cell myelomas: potential clue for diagnosis.

Plasma cell myeloma is a frequent hematogeneous disorder that occurs mainly in older people. Not only bone marrow smears but also clots and/or biopsied specimens are often taken for confirmation of pathological diagnosis. Some specimens show sheet-like plasma cell proliferation associated with immunoglobulin monotype on immunohistology, which readily leads to diagnosis, but many samples do not clearly show light-chain restriction. The aim of the present study was therefore to examine CD79a expression because some samples had reduced expression or none at all. The immunoreactivity of CD79a was categorized into three groups: positive, weakly positive and negative, compared with scattering non-neoplastic plasma cells in the same specimen. Out of 100 specimens of plasma cell myeloma, 48% were positive for CD79a, 15% were weakly positive, and 37% were negative. In contrast, overexpression of cyclinD1 was detected in 26% of examined samples. CD79a-negative cases had a significantly lower percentage of positive staining for cyclinD1 than CD79a-positive or weakly positive cases. Clinicopathological data showed that CD79a-negative expression was associated with decreased platelet numbers in patients. The present study indicates that downregulation or loss of CD79a and/or overexpression of cyclin D1, observed in 59% of neoplastic plasma cell samples, could provide a strong diagnostic clue without regard to the results of immunoglobulin light-chain restriction.

Characterization of c-Maf transcription factor in normal and neoplastic hematolymphoid tissue and its relevance in plasma cell neoplasia.

c-Maf, a leucine zipper-containing transcription factor, is involved in the t(14;16)(q32;q23) translocation found in 5% of myelomas. A causal role for c-Maf in myeloma pathogenesis has been proposed, but data on c-Maf protein expression are lacking. We therefore studied the expression of c-Maf protein by immunohistochemical analysis in myelomas and in a wide variety of hematopoietic tissue. c-Maf protein was detected in a small minority (4.3%) of myelomas, including a t(14;16)(q32;q22-23)/IgH-Maf+ case, suggesting that c-Maf protein is not expressed in the absence of c-Maf rearrangement. In contrast, c-Maf was strongly expressed in hairy cell leukemia (4/4) and in a significant proportion of T-cell (24/42 [57%]) and NK/T-cell (49/97 [51%]) lymphomas, which is in keeping with prior gene expression profiling and transgenic mouse studies. Up-regulation of c-Maf protein occurs in a small subset of myelomas, in hairy cell leukemia, and in T- and NK-cell neoplasms. Its detection may be of particular value in the differential diagnosis of small cell lymphomas.

The costimulatory molecule ICOS regulates the expression of c-Maf and IL-21 in the development of follicular T helper cells and TH-17 cells.

The inducible costimulatory molecule ICOS has been suggested to be important in the development of interleukin 17 (IL-17)-producing helper T cells (T(H)-17 cells) and of follicular helper T cells (T(FH) cells). Here we show that ICOS-deficient mice had no defect in T(H)-17 differentiation but had fewer T(H)-17 cells after IL-23 stimulation and fewer T(FH) cells. We also show that T(FH) cells produced IL-17 and that T(FH) cells in ICOS-deficient mice were defective in IL-17 production. Both T(H)-17 and T(FH) cells had higher expression of the transcription factor c-Maf. Genetic loss of c-Maf resulted in a defect in IL-21 production and fewer T(H)-17 and T(FH) cells. Thus our data suggest that ICOS-induced c-Maf regulates IL-21 production that in turn regulates the expansion of T(H)-17 and T(FH) cells.

Twist-2 controls myeloid lineage development and function.

Basic helix-loop-helix (bHLH) transcription factors play critical roles in lymphoid and erythroid development; however, little is known about their role in myeloid lineage development. In this study, we identify the bHLH transcription factor Twist-2 as a key negative regulator of myeloid lineage development, as manifested by marked increases in mature myeloid populations of macrophages, neutrophils, and basophils in Twist-2-deficient mice. Mechanistic studies demonstrate that Twist-2 inhibits the proliferation as well as differentiation of granulocyte macrophage progenitors (GMP) by interacting with and inhibiting the transcription factors Runx1 and C/EBPalpha. Moreover, Twist-2 was found to have a contrasting effect on cytokine production: inhibiting the production of proinflammatory cytokines such as interleukin-12 (IL-12) and interferon-gamma (IFNgamma) while promoting the regulatory cytokine IL-10 by myeloid cells. The data from further analyses suggest that Twist-2 activates the transcription factor c-Maf, leading to IL-10 expression. In addition, Twist-2 was found to be essential for endotoxin tolerance. Thus, this study reveals the critical role of Twist-2 in regulating the development of myeloid lineages, as well as the function and inflammatory responses of mature myeloid cells.

The initial response of CD4+ IL-4-producing cells.

Naive CD4(+) T cells were reported to produce small amounts of IL-4 in vitro, which are implicated to be sufficient to initiate T(h)2 response in vivo. However, IL-4-producing naive CD4(+) T cells are difficult to study in vivo because they are present in low numbers shortly after the first antigen exposure. Here, we used IL-4/green fluorescence protein (GFP) reporter mice (G4 mice) to track the initial response of CD4(+) IL-4-producing cells. We first established a flow cytometry method to estimate the number of GFP(+) cells. We demonstrated the effectiveness of this method by showing that the responding CD4(+)GFP(+) cells exhibited an activated phenotype, possessed the capacity to express IL-5 and IL-13, but not IFN-gamma mRNA, and showed enhanced levels of GATA3 and c-maf mRNA expression. More importantly, we showed that the cell lines derived from FACS-sorted CD4(+)GFP(+) cells were antigen specific. By using this newly established method, we showed that the majority of responding GFP(+) cells were CD4(+) T cells. Our study provides direct ex vivo evidence to show that a small percent of CD4(+) T cells that have no previous experience of antigenic stimulation might produce IL-4 to initiate T(h)2 response.

Correlation between the expression level of c-maf and glutathione peroxidase-3 in c-maf -/- mice kidney and c-maf overexpressed renal tubular cells.

Large mafs are transcriptional factors and members of the basic leucine zipper (b-Zip) superfamily. Since we previously identified expression of c-maf in mouse kidney, we presently investigated the mRNA expression profile in the kidney of c-maf gene knockout mice by using DNA microarray, and plasma glutathione peroxidase-3 (GPx3) was predominantly downregulated. We focused on the relation between the expression level of c-maf and GPx3 in vivo and in vitro. Since GPx3 is an antioxidant enzyme, oxidative stress was induced by exposing a culture cell derived from mouse renal tubules (mIMCD3) to hydrogen peroxide. Real-time PCR demonstrated that mRNA expression of both c-maf and GPx3 increased in parallel during exposure to oxidative stress in a time- and dose-dependent manner. Then, the mIMCD3 cells were transfected with c-maf-cDNA containing plasmid, which resulted in an increase in mRNA and protein expression of GPx3 compared with the control cells. Thus, c-maf may be transcriptional regulator of GPx3 expression and modulate the antioxidative pathway in the kidney.

Overexpression of c-Maf contributes to T-cell lymphoma in both mice and human.

c-Maf translocation or overexpression has been observed in human multiple myeloma. Although c-maf might function as an oncogene in multiple myeloma, a role for this gene in other cancers has not been shown. In this study, we have found that mice transgenic for c-Maf whose expression was direct to the T-cell compartment developed T-cell lymphoma. Moreover, we showed that cyclin D2, integrin beta(7), and ARK5 were up-regulated in c-Maf transgenic lymphoma cells. Furthermore, 60% of human T-cell lymphomas (11 of 18 cases), classified as angioimmunoblastic T-cell lymphoma, were found to express c-Maf. These results suggest that c-Maf might cause a type of T-cell lymphoma in both mice and humans and that ARK5, in addition to cyclin D2 and integrin beta(7), might be downstream target genes of c-Maf leading to malignant transformation.