Tahseen H. Nasti

Inflammasome Activation of IL-1 Family Mediators in Response to Cutaneous Photodamage†

Although keratinocytes are relatively resistant to ultraviolet radiation (UVR) induced damage, repeated UVR exposure result in accumulated DNA mutations that can lead to epidermal malignancies. Keratinocytes play a central role in elaborating innate responses that lead to inflammation and influence the generation of adaptive immune responses in skin. Apart from the minor cellular constituents of the epidermis, specifically Langerhans cells and melanocytes, keratinocytes are the major source of cytokines. UVR exposure stimulates keratinocytes to secrete abundant pro‐inflammatory IL‐1‐family proteins, IL‐1α, IL‐1β, IL‐18, and IL‐33. Normal skin contains only low levels of inactive precursor forms of IL‐1β and IL‐18, which require caspase 1‐mediated proteolysis for their maturation and secretion. However, caspase‐1 activation is not constitutive, but dependents on the UV‐induced formation of an active inflammasome complex. IL‐1 family cytokines can induce a secondary cascade of mediators and cytokines from keratinocytes and other cells resulting in wide range of innate processes including infiltration of inflammatory leukocytes, induction of immunosuppression, DNA repair or apoptosis. Thus, the ability of keratinocytes to produce a wide repertoire of proinflammatory cytokines can influence the immune response locally as well as systematically, and alter the host response to photodamaged cells. We will highlight differential roles played by each IL‐1 family molecule generated by UV‐damaged keratinocytes, and reveal their complementary influences in modulating acute inflammatory and immunological events that follow cutaneous UV exposure.

Protective Role of Toll-like Receptor 4 during the Initiation Stage of Cutaneous Chemical Carcinogenesis

Toll-like receptors (TLR) activate multiple steps in inflammatory reactions in innate immune responses. They also activate signals that are critically involved in the initiation of adaptive immune responses. Many tumorigenic chemicals have been associated with endotoxin hypersensitivity mediated through TLR4. To determine the role of TLR4 in cutaneous skin carcinogenesis, we treated TLR4-deficient C3H/HeJ mice and the TLR4-normal C3H/HeN mice with the carcinogenic polyaromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA). TLR4-deficient C3H/HeJ mice developed more tumors relative to the TLR4-normal C3H/HeN mice. Both C3H/HeN and C3H/HeJ mice developed a T-cell-mediated immune response to topically applied DMBA. Interestingly, the cell-mediated immune response was mediated by IFN-gamma in C3H/HeN mice and by interleukin (IL)-17 in C3H/HeJ mice. Moreover, C3H/HeN mice had elevated circulating levels of IFN-gamma following topical application of DMBA, whereas IL-17 was elevated in C3H/HeJ mice. The results of this study indicate that TLR4 plays an important role in the prevention of DMBA skin tumorigenesis and that this is associated with differences in the T-cell subtype activated. Efforts to divert the cell-mediated immune response from one that is IL-17 mediated to one that is IFN-gamma mediated may prove to be beneficial in the prevention of DMBA-induced cutaneous tumors.

Heat Shock Proteins HSP27 and HSP70 Are Present in the Skin and Are Important Mediators of Allergic Contact Hypersensitivity

Proteomic analysis of murine skin has shown that a variety of heat shock proteins (HSPs) are constitutively expressed in the skin. Using murine allergic contact hypersensitivity as a model, we investigated the role of two heat shock proteins, HSP27 and HSP70, in the induction of cutaneous cell-mediated immune responses. Immunohistochemical examination of skin specimens showed that HSP27 was present in the epidermis and HSP70 was present in both the epidermis and dermis. Inhibition of HSP27 and HSP70 produced a reduction in the 1-fluoro-2,4-dinitrobenzene contact hypersensitivity response and resulted in the induction of Ag-specific unresponsiveness. Treatment of dendritic cell cultures with recombinant HSP27 caused in the up-regulation of IL-1β, TNF-α, IL-6, IL-12p70, and IL-12p40 but not IL-23p19, which was inhibited when Abs to HSP27 were added. The 1-fluoro-2,4-dinitrobemzene-conjugated dendritic cells that had been treated with HSP27 had an increased capacity to initiate contact hypersensitivity responses compared with control dendritic cells. This augmented capacity required TLR4 signaling because neither cytokine production by dendritic cells nor the increased induction of contact hypersensitivity responses occurred in TLR4-deficient C3H/HeJ mice. Our findings indicate that a cascade of events occurs following initial interaction of hapten with the skin that includes increased activity of HSPs, their interaction with TLR4, and, in turn, increased production of cytokines that are known to enhance Ag presentation by T cells. The results suggest that HSPs form a link between adaptive and innate immunity during the early stages of contact hypersensitivity.

Antagonistic Roles of CD4+ and CD8+ T-Cells in 7,12-Dimethylbenz(a)anthracene Cutaneous Carcinogenesis

The role that cell-mediated immune responses play during cutaneous carcinogenesis has received little attention. In this study, we evaluated the contribution of CD4(+) and CD8(+) T cells in C3H/HeN mice that were subjected to a two-stage 7,12-dimethylbenz(a)anthracene (DMBA) initiation, 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion skin carcinogenesis protocol. In CD8 knockout (CD8(-/-)) mice, allergic contact hypersensitivity to DMBA was reduced compared with wild-type (WT) C3H/HeN mice. On the other hand, CD4 knockout (CD4(-/-)) mice developed an exaggerated contact hypersensitivity response. CD4(+) T cells from DMBA contact-sensitized mice preferentially produced interleukin 4 (IL-4), IL-10, and IL-17; CD8(+) T cells, on the other hand, secreted IFN-gamma. When CD4(-/-), CD8(-/-), and WT mice were subjected to a standard two-stage DMBA/TPA cutaneous carcinogenesis protocol, the percentage of mice with tumors was much greater (P < 0.001) in CD8(-/-) mice than in WT mice. In contrast, the percentage of tumors was significantly less (P < 0.001) in CD4(-/-) mice than in WT mice. Similar results were obtained when the data were evaluated as the number of tumors per mouse. These findings indicate that (a) CD8(+) T cells are the predominant effector cells in allergic contact hypersensitivity to DMBA and that CD4(+) T cells have an inhibitory role and (b) the development of CD8(+) T cells plays a protective role in skin tumor development whereas CD4(+) T cells have the opposite effect. Manipulation of T-cell subpopulations that are induced by carcinogenic chemicals, like DMBA, could be a means of preventing skin cancers caused by these agents.

Resveratrol enhances cell-mediated immune response to DMBA through TLR4 and prevents DMBA induced cutaneous carcinogenesis†

Toll‐like receptors (TLRs) activate signals that are critically involved in innate immune responses and that contribute to the initiation of adaptive immune responses. Resveratrol (trans‐3,5,4‐trihydroxystilbene), a polyphenol found in red grapes and in several other plant sources, is an effective chemopreventive agent in cutaneous chemical carcinogenesis. In this study, we investigated whether TLR4 was required for the chemopreventive action of resveratrol in DMBA skin carcinogenesis. For this purpose, mice with normal and deficient TLR4 function were compared when pretreated with resveratrol and then subjected to a DMBA‐induced skin carcinogenesis protocol. There were fewer tumors/group (P < 0.001) in resveratrol treated TLR4 competent C3H/HeN mice than in TLR4 deficient C3H/HeJ mice. In addition, the size of tumors in C3H/HeN mice was reduced in vivo and their survival in vitro was inhibited by resveratrol to a significantly greater extent than in C3H/HeJ mice. Resveratrol inhibited angiogenesis to a much greater extent in the TLR4 competent mice than in TLR4 deficient mice. IFN‐γ and IL‐12 levels were also increased in TLR4 competent mice compared to TLR4 deficient mice, and TLR4 competent C3H/HeN mice exhibited a greater increase in the cell‐mediated immune response to DMBA. The results of this study indicate that TLR4 is an important mediator of resveratrol chemoprevention in DMBA skin tumorigenesis. Published 2009 Wiley‐Liss, Inc.

Regulation of ultraviolet radiation induced cutaneous photoimmunosuppression by toll-like receptor-4.

UVB radiation is a potent immunosuppressive agent that inhibits cell-mediated immune responses. The mechanisms by which UVB radiation influences cell-mediated immune responses have been the subject of extensive investigation. However, the role of innate immunity on photoimmunological processes has received little attention. The purpose of this study was to determine whether Toll-like receptor-4 (TLR4) contributed to UV-induced suppression of contact hypersensitivity (CHS) responses. TLR4⁻/⁻ and wild type C57BL/6 (TLR4+/+) mice were subjected to a local UVB immunosuppression regimen consisting of 100 mJ/cm² UVB radiation followed by sensitization with the hapten DNFB. Wild type TLR4+/+ mice exhibited significant suppression of contact hypersensitivity response, whereas TLR4⁻/⁻ developed significantly less suppression. The suppression in wild type TLR4+/+ mice could be adoptively transferred to naïve syngeneic recipients. Moreover, there were significantly fewer Foxp3 expressing CD4+CD25+ regulatory T-cells in the draining lymph nodes of UV-irradiated TLR4⁻/⁻ mice than TLR4+/+ mice. When cytokine levels were compared in these two strains after UVB exposure, T-cells from TLR4+/+ mice produced higher levels of IL-10 and TGF-β and lower levels of IFN-γ and IL-17. Strategies to inhibit TLR4 may allow us to develop immunopreventive and immunotherapeutic approaches for management of UVB induced cutaneous immunosuppression.

MC1R, eumelanin and pheomelanin: their role in determining the susceptibility to skin cancer.

Skin pigmentation is due to the accumulation of two types of melanin granules in the keratinocytes. Besides being the most potent blocker of ultraviolet radiation, the role of melanin in photoprotection is complex. This is because one type of melanin called eumelanin is UV absorbent, whereas the other, pheomelanin, is photounstable and may even promote carcinogenesis. Skin hyperpigmentation may be caused by stress or exposure to sunlight, which stimulates the release of α‐melanocyte stimulating hormone (α‐MSH) from damaged keratinocytes. Melanocortin 1 receptor (MC1R) is a key signaling molecule on melanocytes that responds to α‐MSH by inducing expression of enzymes responsible for eumelanin synthesis. Persons with red hair have mutations in the MC1R causing its inactivation; this leads to a paucity of eumelanin production and makes red‐heads more susceptible to skin cancer. Apart from its effects on melanin production, the α‐MSH/MC1R signaling is also a potent anti‐inflammatory pathway and has been shown to promote antimelanoma immunity. This review will focus on the role of MC1R in terms of its regulation of melanogenesis and influence on the immune system with respect to skin cancer susceptibility.

Cell mediated immune responses through TLR4 prevents DMBA‐induced mammary carcinogenesis in mice

Toll‐like receptors (TLRs) activate signals that are critically involved in the initiation of adaptive immune responses and many tumorigenic chemicals have been associated with activation of those pathways. To determine the role of TLR‐4 (TLR4) in mammary carcinogenesis, we subjected TLR4 deficient and wild type (WT) mice to oral gavage with carcinogenic polyaromatic hydrocarbon 7,12‐dimethylbenz(a)anthracene (DMBA). TLR4 deficient mice developed more tumors relative to the WT mice. T cells of TLR4 deficient mice produced elevated levels of IL‐17 and lower levels of IFN‐γ relative to WT mice. IL‐12 secreted by CD11c+ cells was higher in WT mice, whereas greater amounts of IL‐23 were produced by CD11c+ cells from TLR4 deficient mice. Moreover, there was higher incidence of regulatory T cells in TLR4 deficient mice than WT mice. Similarly, various markers of angiogenesis [matrix metalloproteinases (MMP)‐2 and MMP‐9, CD31 and vascular endothelial growth factor] were highly expressed in tumors from TLR4 deficient mice than WT mice. The results of this study indicate that TLR4 plays an important role in the prevention of DMBA induced mouse mammary tumorigenesis and efforts to divert the cell‐mediated immune response may, therefore, prove to be beneficial in the prevention of mammary tumors.

Differential roles of T-cell subsets in regulation of ultraviolet radiation induced cutaneous photocarcinogenesis.

Ultraviolet (UV) radiation, in particular the midwavelength range (UVB; 290–320 nm), is one of the most significant risk factors for the development of nonmelanoma skin cancer. UVB radiation‐induced immunosuppression, which occurs in both humans and laboratory animals, contributes to their pathogenesis. However, there are conflicting reports on the relative role of CD4+ and CD8+ T cells in UVB induced skin cancer. The purpose of this study was to delineate the contribution of these two cell subpopulations to UVB induced immunosuppression and tumor development using C3H/HeN (WT), CD4 knockout (CD4−/−) and CD8 knockout (CD8−/−) mice. We observed that UVB induced skin carcinogenesis was retarded in terms of number of tumors per group, tumor volume and percentage of mice with tumors, in mice deficient in CD4+ T cells compared with wild‐type mice, whereas significantly greater (P < 0.05) numbers of tumors occurred in CD8−/− mice. These results indicate that, CD4+ T cells promote tumor development while CD8+ T cells have the opposite effect. Further, we found that CD4+ T cells from tumor‐bearing mice produced interleukin (IL)‐4, IL‐10, and IL‐17 whereas CD8+ T cells produced interferon‐γ. Manipulation of T‐cell subpopulations that are induced by UVB radiation could be a means of preventing skin cancers caused by this agent.

The antiproliferative function of violacein-like purple violet pigment (PVP) from an Antarctic Janthinobacterium sp. Ant5-2 in UV-induced 2237 fibrosarcoma.

Background  In this study, we have investigated the chemotherapeutic potential of a purple violet pigment (PVP), which was isolated from a previously undescribed Antarctic Janthinobacterium sp. (Ant5‐2), against murine UV‐induced 2237 fibrosarcoma and B16F10 melanoma cells.