NF-κB Inhibition through Proteasome Inhibition or IKKβ Blockade Increases the Susceptibility of Melanoma Cells to Cytostatic Treatment through Distinct Pathways

Metastasized melanoma is almost universally resistant to chemotherapy. Given that constitutive or drug-induced upregulation of NF-κB activity is associated with this chemoresistance, NF-κB inhibition may increase the susceptibility to antitumoral therapy. On the cellular level, two principles of NF-κB inhibition, proteasome inhibition by bortezomib and IκB kinase-β (IKKβ) inhibition by the kinase inhibitor of NF-κB-1 (KINK-1), significantly increased the antitumoral efficacy of camptothecin. When combined with camptothecin, either of the two NF-κB-inhibiting principles synergistically influenced progression-related in vitro functions, including cell growth, apoptosis, and invasion through an artificial basement membrane. In addition, when C57BL/6 mice were intravenously injected with B16F10 melanoma cells, the combination of cytostatic treatment with either of the NF-κB-inhibiting compounds revealed significantly reduced pulmonary metastasis compared to either treatment alone. However, on the molecular level, nuclear translocation of p65, cell cycle analysis, and expression of NF-κB-dependent gene products disclosed distinctly different molecular mechanisms, resulting in the same functional effect. That proteasome inhibition and IKKβ inhibition affect distinct molecular pathways downstream of NF-κB, both leading to increased chemosensitivity, is previously unreported. Thus, it is conceivable that switching the two principles of NF-κB inhibition, once resistance to one of the agents occurs, will improve future treatment regimens

Involvement of IL-9 in Th17-Associated Inflammation and Angiogenesis of Psoriasis

<div><p>It is thought that a Th1/Th17-weighted immune response plays a predominant role in the pathogenesis of psoriasis. Our findings now indicate a link between IL-9, a Th2 and Th9 cytokine, and Th17 pathway in psoriasis. In K5.hTGF-β1 transgenic mice, exhibiting a psoriasis-like phenotype, we found increased IL-9R and IL-9 expression in the skin and intradermal IL-9 injection induced Th17-related inflammation. IL-9 also promoted angiogenesis and VEGF and CD31 overexpression in mice <em>in vivo</em> and increased tube formation of human endothelial cells <em>in vitro</em>. Injecting anti-IL-9 antibody into K5.hTGF-β1 transgenic mice not only diminished inflammation (including skin infiltration by T cells, monocytes/macrophages, and mast cells) and angiogenesis but also delayed the psoriasis-like skin phenotype. Notably, injection of anti-psoriatic acting anti-IL-17 antibody reduced skin IL-9 mRNA and serum IL-9 protein levels in K5.hTGF-β1 transgenic mice and prevented IL-9-induced epidermal hyperplasia and inflammation of the skin of wild type mice. In addition, we observed that IL-9R expression in lesional skin from psoriasis patients was markedly higher than in healthy skin from control subjects. Moreover, IL-9 significantly enhanced IL-17A production by cultured human peripheral blood mononuclear cells or CD4+ T cells, especially in psoriasis patients. Thus, IL-9 may play a role in the development of psoriatic lesions through Th17-associated inflammation and angiogenesis.</p> </div

IL-9 induces angiogenesis in mice and tube formation in HDMEC.

<p>(<b>A</b>) Representative photomicrographs of VEGF and CD31 staining of paraffin-embedded sections of skin from WT or K5.hTGF-β1 transgenic mice injected intradermally for 4 days with 500 ng of IL-9 or vehicle (PBS) (Scale bar 200 µm). (<b>B,C</b>) Semi-quantitative scoring of VEGF and CD31 positivity (n = 4 mice per group). (<b>D</b>) <i>In vitro</i> angiogenesis assay (tube formation) was performed with human dermal micro vascular endothelial cells (HDMEC) in the presence or absence of IL-9 (scale bars 100 µm). (<b>E</b>) Bifurcations were counted as a measure of blood vessel formation. Data are from one of two independent experiments (n = 5 random fields in each sample). Error bars represent SEM. *, p)0.05; ***, p)0.001. (unpaired t-test). Similar results were obtained in three independent experiments.</p

IL-9 accelerates psoriasis-like inflammation in K5.hTGF-β1 transgenic mice.

<p>(<b>A</b>) Representative photomicrographs of immunohistochemical staining of IL-9 and IL-9R in the dorsal skin of K5.hTGF-β1 transgenic mice (Scale bar 200 µm for WT and 50 and 100 µm for K5.hTGF-β1 epidermis and dermis, respectively). (<b>B</b>) Real time PCR analysis for IL-9 in the dorsal skin of WT and K5.hTGF-β1 transgenic mice (n = 7 mice per group). (<b>C</b>) K5.hTGF-β1 transgenic mice were injected intradermally for 4 days with 500 ng of IL-9 or vehicle (PBS) and skin samples were collected 24 hours after the last IL-9 injection (C,D). Representative photomicrographs of HE-stained paraffin-embedded skin sections (Scale bar 200 µm). Histological quantification of mean epidermal thickness (n = 5 mice per group). (<b>D</b>) Dermal infiltration by CD3+ T cells, CD68+ monocytes/macrophages, and mast cells in the dorsal skin of WT mice (n = 5 mice per group). Data shown represent mean numbers of cells per ×200 microscopic field. Error bars represent SEM. *, p)0.05 (unpaired t-test). Similar results were obtained in two independent experiments.</p

IL-9 enhances IL-17A production in human psoriasis.

<p>(<b>A</b>) Immunohistochemical staining of IL-9R in normal and lesional human psoriatic skin. (Scale bar 100 µm for normal skin and 50 µm for psoriatic skin). (<b>B</b>) Number of IL-9R positive cells in lesional skin from psoriatic patients or healthy skin from normal control subjects (n = 3 subjects per group). (<b>C</b>) Detection of IL-9 protein level in the culture supernatant of activated and cultured CD4+ T cells of healthy or psoriatic human subjects by ELISA (n = 4 subjects per group). (<b>D</b>) Detection of IL-17A in the culture supernatant of activated and cultured PBMC and CD4+ T cells of healthy or psoriatic human subjects as determined by ELISA. Cells were stimulated with IL-9 or left unstimulated (n = 3–4 subjects per group). (<b>E</b>) Dual ELIspot assay for IL-17 and IFN-γ of activated and cultured CD4+ T cells of psoriatic subjects in the presence or absence of IL-9. Number of CD4+ T cells secreting IL-17 or IFN-γ, or co-secreting IL-17 and IFN-γ were counted in samples from psoriatic patients (n = 4). (<b>F</b>) Detection of IL-17A in activated and cultured CD4+ T cells of psoriatic patients by ELISA. Cells were stimulated with different cytokine combinations (n = 4 subjects per group). Error bars represent SEM. *, p)0.05; **, p)0.01; ***, p)0.001. (unpaired t-test). ND, not detected.</p

Anti-IL-9 therapy inhibits the psoriatic skin phenotype, inflammation and angiogenesis in K5.hTGF-β1 transgenic mice.

<p>K5.hTGF-β1 transgenic mice were injected i.p. with either anti-IL-9 antibody or IgG isotype control antibody (10 mg/kg) twice a week for 4 weeks and skin samples were collected at the end of week 4 for analysis (n = 5 mice per treatment group). WT mice served as controls. (<b>A</b>) Mean diseases severity scores for anti-IL9- vs. IgG-treated mice. (<b>B</b>) Representative photomicrographs of HE-stained paraffin-embedded sections of the skin from K5.hTGF-β1 transgenic mice at the end of week 4 of treatment vs. skin of an untreated WT mouse (Scale bar 200 um). (<b>C</b>) Epidermal thickness and dermal infiltration cells by CD3+ T cells, CD68+ monocytes/macrophages, and mast cells (mean numbers per ×200 microscopic field) in the dorsal skin of K5.hTGF-β1 transgenic mice. (<b>D</b>) Real-time PCR analysis of IL-17A, STAT3, and IFN-γ in the dorsal skin of K5.hTGF-β1 transgenic mice. (<b>E</b>) Representative photomicrographs of VEGF and CD31 staining of paraffin-embedded sections of skin from K5.hTGF-β1 transgenic mice (Scale bar 200 um). (<b>F</b>) Semi-quantitative scoring of VEGF and CD31 positivity. Error bars represent SEM. *, p)0.05; **, p)0.01. (Uunpaired t-test).</p

8-Methoxypsoralen Plus Ultraviolet A Therapy Acts via Inhibition of the IL-23/Th17 Axis and Induction of Foxp3(+) Regulatory T Cells Involving CTLA4 Signaling in a Psoriasis-Like Skin Disorder

To elucidate the molecular action of 8-methoxypsoralen plus UVA (PUVA), a standard dermatological therapy, we used K5. hTGF-beta 1 transgenic mice exhibiting a skin phenotype and cytokine abnormalities with strong similarities to human psoriasis. We observed that impaired function of CD4(+)CD25(+) regulatory T cells (Tregs) and increased cytokine levels of the IL-23/Th17 pathway were responsible for the psoriatic phenotype in this mouse model. Treatment of K5.hTGF-beta 1 transgenic mice with PUVA suppressed the IL-23/Th17 pathway, Th1 milieu, as well as transcription factors STAT3 and orphan nuclear receptor ROR gamma t. PUVA induced the Th2 pathway and IL-10-producing CD4(+)CD25(+)Foxp3(+)Tregs with disease-suppressive activity that was abolished by anti-CTLA4 mAb treatment. These findings were paralleled by macroscopic and microscopic clearance of the diseased murine skin. Anti-IL-17 mAb treatment also diminished the psoriatic phenotype of the mice. This indicated that both induced Tregs involving CTLA4 signaling and inhibition of the IL-23/Th17 axis are central for the therapeutic action of PUVA. The Journal of Immunology, 2010, 184: 7257-7267

Halting angiogenesis by non-viral somatic gene therapy alleviates psoriasis and murine psoriasiform skin lesions

Dysregulated angiogenesis is a hallmark of chronic inflammatory diseases, including psoriasis, a common skin disorder that affects approximately 2% of the population. Studying both human psoriasis in 2 complementary xenotransplantation models and psoriasis-like skin lesions in transgenic mice with epidermal expression of human TGF-β1, we have demonstrated that antiangiogenic non-viral somatic gene therapy reduces the cutaneous microvasculature and alleviates chronic inflammatory skin disorders. Transient muscular expression of the recombinant disintegrin domain (RDD) of metargidin (also known as ADAM-15) by in vivo electroporation reduced cutaneous angiogenesis and vascularization in all 3 models. As demonstrated using red fluorescent protein–coupled RDD, the treatment resulted in muscular expression of the gene product and its deposition within the cutaneous hyperangiogenic connective tissue. High-resolution ultrasound revealed reduced cutaneous blood flow in vivo after electroporation with RDD but not with control plasmids. In addition, angiogenesis- and inflammation-related molecular markers, keratinocyte proliferation, epidermal thickness, and clinical disease scores were downregulated in all models. Thus, non-viral antiangiogenic gene therapy can alleviate psoriasis and may do so in other angiogenesis-related inflammatory skin disorders