Interferon-γ links ultraviolet radiation to melanomagenesis in mice.

Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, the incidence of which continues to rise. Epidemiological studies show that the major aetiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood. We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and aetiological criteria, but only when irradiated as neonatal pups with UVB, not UVA. However, the mechanisms underlying UVB-initiated, neonatal-specific melanomagenesis remain largely unknown. Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-γ (IFN-γ), but not type-I interferons. IFN-γ was produced by macrophages recruited to neonatal skin by UVB-induced ligands to the chemokine receptor Ccr2. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-γ blockade abolished macrophage-enhanced melanoma growth and survival. IFN-γ-producing macrophages were also identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-γ in promoting melanocytic cell survival/immunoevasion, identifying a novel candidate therapeutic target for a subset of melanoma patients.

LAB/NTAL facilitates fungal/PAMP-induced IL-12 and IFN-γ production by repressing ß-catenin activation in dendritic cells.

Fungal pathogens elicit cytokine responses downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled or hemiITAM-containing receptors and TLRs. The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses. Here we demonstrate that LAB is involved in anti-fungal immunity. We show that Lat2-/- mice are more susceptible to C. albicans infection than wild type (WT) mice. Dendritic cells (DCs) express LAB and we show that it is basally phosphorylated by the growth factor M-CSF or following engagement of Dectin-2, but not Dectin-1. Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear ß-catenin levels. This in turn is important for controlling fungal/PAMP-induced cytokine production in DCs. C. albicans- and LPS-induced IL-12 and IL-23 production was blunted in Lat2-/- DCs. Accordingly, Lat2-/- DCs directed reduced Th1 polarization in vitro and Lat2-/- mice displayed reduced Natural Killer (NK) and T cell-mediated IFN-γ production in vivo/ex vivo. Thus our data define a novel link between LAB and ß-catenin nuclear accumulation in DCs that facilitates IFN-γ responses during anti-fungal immunity. In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance.

Regulation of interleukin-12/interleukin-23 production and the T-helper 17 response in humans.

Interleukin-12 (IL-12) and IL-23 share a common chain. Yet, their production in response to pathogens is differentially regulated, and their functions are distinct and often antithetic. IL-12 is involved in the induction or amplification of the T-helper (Th) type 1 response, whereas IL-23 has been associated with the generation of the Th17 response and IL-17 production. Mycobacterium tuberculosis and yeast zymosan induce IL-23, but in the absence of other stimuli, no IL-12 is induced in human dendritic cells (DCs). The stimulation of IL-23 by M. tuberculosis was mostly explained by the triggering of Toll-like receptor (TLR2) and the cytoplasmic receptor nucleotide oligomerization domain (NOD)-containing protein 2, whereas zymosan induces IL-23 primarily by stimulating the beta-glucan receptor dectin-1 alone or in combination with TLR2. IL-23, IL-6, transforming growth factor (TGF-beta1), and IL-1beta in supernatants from activated human DCs induce human naive CD4(+) T cells to produce IL-17. These data are consistent with various recent reports that TGF-beta is an inducer of IL-17 production both in human and in mouse cells. However, IL-1 is necessary in combination with some or all of the other cytokines to induce IL-17 production in human T cells. The ability of various stimuli to induce Th17 cells depends not only on their induction of IL-23, IL-6, and TGF-beta production in DCs but also on their ability to activate directly or indirectly the inflammasome and to induce IL-1beta.

T-Cell Deletion of MyD88 Connects IL17 and IκBζ to RAS Oncogenesis.

Cancer development requires a favorable tissue microenvironment. By deleting Myd88 in keratinocytes or specific bone marrow subpopulations in oncogenic RAS-mediated skin carcinogenesis, we show that IL17 from infiltrating T cells and IκBζ signaling in keratinocytes are essential to produce a permissive microenvironment and tumor formation. Both normal and RAS-transformed keratinocytes respond to tumor promoters by activating canonical NF-κB and IκBζ signaling, releasing specific cytokines and chemokines that attract Th17 cells through MyD88-dependent signaling in T cells. The release of IL17 into the microenvironment elevates IκBζ in normal and RAS-transformed keratinocytes. Activation of IκBζ signaling is required for the expression of specific promoting factors induced by IL17 in normal keratinocytes and constitutively expressed in RAS-initiated keratinocytes. Deletion of Nfkbiz in keratinocytes impairs RAS-mediated benign tumor formation. Transcriptional profiling and gene set enrichment analysis of IκBζ-deficient RAS-initiated keratinocytes indicate that IκBζ signaling is common for RAS transformation of multiple epithelial cancers. Probing The Cancer Genome Atlas datasets using this transcriptional profile indicates that reduction of IκBζ signaling during cancer progression associates with poor prognosis in RAS-driven human cancers. IMPLICATIONS: The paradox that elevation of IκBζ and stimulation of IκBζ signaling through tumor extrinsic factors is required for RAS-mediated benign tumor formation while relative IκBζ expression is reduced in advanced cancers with poor prognosis implies that tumor cells switch from microenvironmental dependency early in carcinogenesis to cell-autonomous pathways during cancer progression.

Selenium compounds inhibit I kappa B kinase (IKK) and nuclear factor-kappa B (NF-kappa B) in prostate cancer cells.

Selenium compounds are potential chemopreventive agents for prostate cancer. There are several proposed mechanisms for their anticancer effect, including enhanced apoptosis of transformed cells. Because the transcription factor nuclear factor-kappa B (NF-kappa B) is often constitutively activated in tumors and is a key antiapoptotic factor in mammalian cells, we tested whether selenium inhibited NF-kappa B activity in prostate cancer cells. In our work, we used sodium selenite and a novel synthetic compound, methylseleninic acid (MSeA), that served as a precursor of the putative active monomethyl metabolite methylselenol. We found that both selenium forms inhibited cell growth and induced apoptosis in DU145 and JCA1 prostate carcinoma cells. Sodium selenite and MeSeA, at the concentrations that induced apoptosis, inhibited NF-kappa B DNA binding induced by tumor necrosis factor-alpha and lipopolysaccharide in DU145 and JCA1 prostate cells. Both compounds also inhibited kappa B. Luciferase reporter activity in prostate cells. A key to NF-kappa B regulation is the inhibitory kappa B (I kappa B) proteins that in response to diverse stimuli are rapidly phosphorylated by I kappa B kinase complex, ubiquitinated, and undergo degradation, releasing NF-kappa B factor. We showed that sodium selenite and MSeA inhibited I kappa B kinase activation and I kappa B-alpha phosphorylation and degradation induced by TNF-alpha and lipopolysaccharide in prostate cells. NF-kappa B blockage by I kappa B-alpha d.n. mutant resulted in the sensitization of prostate carcinoma cells to apoptosis induced by selenium compounds. These results suggest that selenium may target the NF-kappa B activation pathway to exert, at least in part, its cancer chemopreventive effect in prostate.

Interleukin-1 and interferon-γ orchestrate ß-glucan-activated human dendritic cell programming via IκB-ζ modulation.

Recognition of microbial components via innate receptors including the C-type lectin receptor Dectin-1, together with the inflammatory environment, programs dendritic cells (DCs) to orchestrate the magnitude and type of adaptive immune responses. The exposure to ß-glucan, a known Dectin-1 agonist and component of fungi, yeasts, and certain immune support supplements, activates DCs to induce T helper (Th)17 cells that are essential against fungal pathogens and extracellular bacteria but may trigger inflammatory pathology or autoimmune diseases. However, the exact mechanisms of DC programming by ß-glucan have not yet been fully elucidated. Using a gene expression/perturbation approach, we demonstrate that in human DCs ß-glucan transcriptionally activates via an interleukin (IL)-1- and inflammasome-mediated positive feedback late-induced genes that bridge innate and adaptive immunity. We report that in addition to its known ability to directly prime T cells toward the Th17 lineage, IL-1 by promoting the transcriptional cofactor inhibitor of κB-ζ (IκB-ζ) also programs ß-glucan-exposed DCs to express cell adhesion and migration mediators, antimicrobial molecules, and Th17-polarizing factors. Interferon (IFN)-γ interferes with the IL-1/IκB-ζ axis in ß-glucan-activated DCs and promotes T cell-mediated immune responses with increased release of IFN-γ and IL-22, and diminished production of IL-17. Thus, our results identify IL-1 and IFN-γ as regulators of DC programming by ß-glucan. These molecular networks provide new insights into the regulation of the Th17 response as well as new targets for the modulation of immune responses to ß-glucan-containing microorganisms.

High expression of SNIP1 correlates with poor prognosis in non-small cell lung cancer and SNIP1 interferes with the recruitment of HDAC1 to RB in vitro.

The Rb tumor suppressor gene performs a critical role in controlling cell proliferation and tumorigenesis; it recruits HDAC1 protein into the E2F complexes to repress transcription. In this study, we demonstrate that SNIP1, RB and HDAC1 were significantly expressed in same lung cancer tissues in a tissue microarray (TMA) containing 300 non-small cell lung cancers (NSCLC). High expression level of SNIP1 in tumor patients was significantly correlated with poor prognosis in NSCLC (log-rank P for OS = 0.01, log-rank P for DFS = 0.001). Functionally, SNIP1 competes with HDAC1 for binding to RB and reduces HDAC activity in vitro. Knockdown of SNIP1 reduced colony formation ability of lung cancer cells. These findings may indicate the involvement of SNIP1 in progression of lung cancer by regulating the RB/HDAC1 interaction.

IL-1R-MyD88 signaling in keratinocyte transformation and carcinogenesis.

Constitutively active RAS plays a central role in the development of human cancer and is sufficient to induce tumors in two-stage skin carcinogenesis. RAS-mediated tumor formation is commonly associated with up-regulation of cytokines and chemokines that mediate an inflammatory response considered relevant to oncogenesis. In this study, we report that mice lacking IL-1R or MyD88 are less sensitive to topical skin carcinogenesis than their respective wild-type (WT) controls. MyD88(-/-) or IL-1R(-/-) keratinocytes expressing oncogenic RAS are hyperproliferative and fail to up-regulate proinflammatory genes or down-regulate differentiation markers characteristic of RAS-expressing WT keratinocytes. Although RAS-expressing MyD88(-/-) keratinocytes form only a few small tumors in orthotopic grafts, IL-1R-deficient RAS-expressing keratinocytes retain the ability to form tumors in orthotopic grafts. Using both genetic and pharmacological approaches, we find that the differentiation and proinflammatory effects of oncogenic RAS in keratinocytes require the establishment of an autocrine loop through IL-1α, IL-1R, and MyD88 leading to phosphorylation of IκBα and NF-κB activation. Blocking IL-1α-mediated NF-κB activation in RAS-expressing WT keratinocytes reverses the differentiation defect and inhibits proinflammatory gene expression. Collectively, these results demonstrate that MyD88 exerts a cell-intrinsic function in RAS-mediated transformation of keratinocytes.

Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells.

We analyzed interleukin (IL) 12 and IL-23 production by monocyte-derived dendritic cells (mono-DCs). Mycobacterium tuberculosis H37Rv and zymosan preferentially induced IL-23. IL-23 but not IL-12 was efficiently induced by the combination of nucleotide-binding oligodimerization domain and Toll-like receptor (TLR) 2 ligands, which mimics activation by M. tuberculosis, or by the human dectin-1 ligand beta-glucan alone or in combination with TLR2 ligands, mimicking induction by zymosan. TLR2 ligands inhibited IL-12 and increased IL-23 production. DC priming with interferon (IFN) gamma strongly increased IL-12 production, but was not required for IL-23 production and inhibited IL-23 production induced by beta-glucan. The pattern of IL-12 and IL-23 induction was reflected in accumulation of the IL-12p35 and IL-23p19 transcripts, respectively, but not IL-12/23p40. Although IL-23, transforming growth factor beta, and IL-6 contained in the supernatants of activated mono-DCs played a role in the induction of IL-17 by human CD4(+) T cells, IL-1beta, in combination with one or more of those factors, was required for IL-17 production, and its production determined the differential ability of the stimuli used to elicit mono-DCs to produce soluble factors directing IL-17 production. Thus, the differential ability of pathogens to induce antigen-presenting cells to produce cytokines regulates the immune response to infection.

SNIP1 is a candidate modifier of the transcriptional activity of c-Myc on E box-dependent target genes.

Using a yeast two-hybrid screen, we found that SNIP1 (Smad nuclear-interacting protein 1) associates with c-Myc, a key regulator of cell proliferation and transformation. We demonstrate that SNIP1 functions as an important regulator of c-Myc activity, binding the N terminus of c-Myc through its own C terminus, and that SNIP1 enhances the transcriptional activity of c-Myc both by stabilizing it against proteosomal degradation and by bridging the c-Myc/p300 complex. These effects of SNIP1 on c-Myc likely contribute to synergistic effects of SNIP1, c-Myc, and H-Ras in inducing formation of foci in an in vitro transformation assay and also in supporting anchorage-independent growth. The significant association of SNIP1 and c-Myc staining in a non-small cell lung cancer tissue array is further evidence that their activities might be linked and suggests that SNIP1 might be an important modulator of c-Myc activity in carcinogenesis.

TGF-beta and vitamin D3 utilize distinct pathways to suppress IL-12 production and modulate rapid differentiation of human monocytes into CD83+ dendritic cells.

We previously demonstrated that agents known to signal infection or inflammation can rapidly and directly drive differentiation of human CD14+ monocytes into CD83+ dendritic cells (DCs) when introduced to cells under serum-free conditions. In this study, we evaluated the effects of TGF-beta and vitamin D3 (VitD3) on the proportion and function of monocytes that adopt DC characteristics. TGF-beta significantly decreased the proportion of cells that rapidly adopted stable DC characteristics in response to LPS, but had little or no effect on calcium ionophore-induced differentiation. In contrast, VitD3 showed no such pathway specificity and dramatically suppressed differentiation of monocytes into DCs in response to these agents. Both TGF-beta and VitD3 altered cytokine and chemokine production in LPS-treated monocytes, inhibited IL-12 and IL-10 secretion, and decreased the functional capacity of DCs. Despite the similar effects of TGF-beta and VitD3, there are significant differences in the signaling pathways used by these agents, as evidenced by their distinct effects on LPS- and calcium ionophore-induced DC differentiation, on LPS-induced secretion of IL-10, and on two members of the NF-kappaB family of transcription factors, RelB and cRel. These studies identify TGF-beta and VitD3 as potent regulatory factors that use distinct pathways to suppress both the differentiation of DCs as well as their capacity to secrete the Th1-polarizing cytokine IL-12. Because these agents are present in serum and negatively affect DC differentiation at physiological concentrations, our findings are likely to have significance regarding the in vivo role of TGF-beta and VitD3 in determining the type of immune responses.

Adenovirus type 5 vectors induce dendritic cell differentiation in human CD14(+) monocytes cultured under serum-free conditions.

To determine whether infection by a model virus is capable of initiating dendritic cell (DC) differentiation, human CD14(+) peripheral blood monocytes were infected with replication-defective type 5 adenovirus. Under serum-free conditions, this resulted in differentiation of a majority of cells toward a DC phenotype within 36 to 48 hours, without the need for cytokine-induced predifferentiation. Infection induced DC morphology and altered the expression of surface markers, including loss of CD14, de novo induction of CD83 and CD25, and strongly augmented expression of CD86, CD80, CD40, and HLA-DR and HLA class I molecules. Differentiated cells maintained immunophenotype without loss of viability for at least 2 days after removal of the differentiation agent and cytokines. A greatly enhanced capacity to stimulate T-lymphocyte alloproliferation and increased expression of the DC-associated transcription factor RelB were observed. Virus without transgene was found to induce changes similar to transgene-expressing viruses. RelB up-regulation and DC immunophenotype were sensitive to the antioxidant N-acetylcysteine, suggesting a critical role for nuclear factor kappaB. RNAse protection assays revealed elevated levels of messenger RNA for a number of chemokines and cytokines associated with DCs. Finally, during differentiation, adenovirus-infected monocytes were shown to secrete chemokines and cytokines, including tumor necrosis factor-alpha (TNF-alpha). Furthermore, a TNF-alpha-neutralizing antibody inhibited the expression of some DC surface markers, indicating a contributing role for this cytokine in the adenovirus-induced differentiation of DC from monocytes. These findings have implications for the biology of monocytes as precursors to DCs and also for the use of recombinant adenovirus in vaccines or gene therapy.

Role of Smad3 in the hormonal modulation of in vivo wound healing responses.

Smad3 is involved in mediating intracellular signaling by members of the transforming growth factor-beta superfamily and plays a critical role in the cellular proliferation, differentiation, migration, and elaboration of matrix pivotal to cutaneous wound healing. Cross-talk between Smad3 and hormone signaling in vitro has been suggested as an important control mechanism regulating cell activities; however, its relevance in vivo is unknown. Here we report that Smad3 plays a role in androgen-mediated inhibition of wound healing but not in the responses to estrogen modulation in vivo. Both wild-type and Smad3 null female mice exhibited delayed healing following ovariectomy, which could be reversed by estrogen replacement. By contrast, castration accelerated healing in wild-type male mice and was reversible by exogenous androgen treatment. Intriguingly, modulation of androgen levels resulted in no discernible perturbation in the healing response in the Smad3 null mice. Mutant monocytes could be lipopolysaccharide stimulated to produce specific pro-inflammatory agents (macrophage monocyte inhibitory factor) in a fashion similar to wild-type cells, but exhibited a muted response to androgen-mediated stimulation while maintaining a normal response to estrogen-induced macrophage inhibitory factor inhibition. These data suggest that Smad3 plays a role in mediating androgen signaling during the normal wound healing response and implicate Smad3 in the modulation of inflammatory cell activity by androgens.

Levels of phospho-Smad2/3 are sensors of the interplay between effects of TGF-beta and retinoic acid on monocytic and granulocytic differentiation of HL-60 cells.

We have investigated the role of Smad family proteins, known to be important cytoplasmic mediators of signals from the transforming growth factor-beta (TGF-beta) receptor serine/threonine kinases, in TGF-beta-dependent differentiation of hematopoietic cells, using as a model the human promyelocytic leukemia cell line, HL-60. TGF-beta-dependent differentiation of these cells to monocytes, but not retinoic acid-dependent differentiation to granulocytes, was accompanied by rapid phosphorylation and nuclear translocation of Smad2 and Smad3. Vitamin D(3) also induced phosphorylation of Smad2/3 and monocytic differentiation; however the effects were indirect, dependent on its ability to induce expression of TGF-beta1. Simultaneous treatment of these cells with TGF-beta1 and all-trans-retinoic acid (ATRA), which leads to almost equal numbers of granulocytes and monocytes, significantly reduced the level of phospho-Smad2/3 and its nuclear accumulation, compared with that in cells treated with TGF-beta1 alone. TGF-beta1 and ATRA activate P42/44 mitogen-activated protein (MAP) kinase with nearly identical kinetics, ruling out its involvement in these effects on Smad phosphorylation. Addition of the inhibitor-of-protein serine/threonine phosphatases, okadaic acid, blocks the ATRA-mediated reduction in TGF-beta-induced phospho-Smad2 and shifts the differentiation toward monocytic end points. In HL-60R mutant cells, which harbor a defective retinoic acid receptor-alpha (RAR-alpha), ATRA is unable to reduce levels of TGF-beta-induced phospho-Smad2/3, coincident with its inability to differentiate these cells along granulocytic pathways. Together, these data suggest a new level of cross-talk between ATRA and TGF-beta, whereby a putative RAR-alpha-dependent phosphatase activity limits the levels of phospho-Smad2/3 induced by TGF-beta, ultimately reducing the levels of nuclear Smad complexes mediating the TGF-beta-dependent differentiation of the cells to monocytic end points.