STING-Dependent Interferon-λ1 Induction in HT29 Cells, a Human Colorectal Cancer Cell Line, After Gamma-Radiation.

PURPOSE: To investigate the induction of type III interferons (IFNs) in human cancer cells by gamma-rays. METHODS AND MATERIALS: Type III IFN expression in human cancer cell lines after gamma-ray irradiation in vitro was assessed by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Signaling pathways mediating type III IFN induction were examined by a variety of means, including immunoblotting, flow cytometry, confocal imaging, and reverse transcription-quantitative polymerase chain reaction. Key mediators in these pathways were further explored and validated using gene CRISPR knockout or short hairpin RNA knockdown. RESULTS: Exposure to gamma-rays directly induced type III IFNs (mainly IFNL1) in human cancer cell lines in dose- and time-dependent fashions. The induction of IFNL1 was primarily mediated by the cytosolic DNA sensors-STING-TBK1-IRF1 signaling axis, with a lesser contribution from the nuclear factor kappa b signaling in HT29 cells. In addition, type III IFN signaling through its receptors serves as a positive feedback loop, further enhancing IFN expression via up-regulation of the kinases in the STING-TBK1 signaling axis. CONCLUSIONS: Our results suggest that IFNL1 can be up-regulated in human cancer cell lines after gamma-ray treatment. In HT29 cells this induction occurs via the STING pathway, adding another layer of complexity to the understanding of radiation-induced antitumor immunity, and may provide novel insights into IFN-based cancer treatment.

Langerhans Cells Facilitate UVB-Induced Epidermal Carcinogenesis.

UVB light is considered the major environmental inducer of human keratinocyte (KC) DNA mutations, including within the tumor-suppressor gene p53, and chronic exposure is associated with cutaneous squamous cell carcinoma formation. Langerhans cells (LCs) comprise a dendritic network within the suprabasilar epidermis, yet the role of LCs in UVB-induced carcinogenesis is largely unknown. Herein we show that LC-intact epidermis develops UVB-induced tumors more readily than LC-deficient epidermis. Although levels of epidermal cyclopyrimidine dimers following acute UVB exposure are equivalent in the presence or absence of LCs, chronic UVB-induced p53 mutant clonal islands expand more readily in association with LCs, which remain largely intact and are preferentially found in proximity to the expanding mutant KC populations. The observed LC facilitation of mutant p53 clonal expansion is completely αß and γδ T-cell independent and is associated with increased intraepidermal expression of IL-22 and the presence of group 3 innate lymphoid cells. These data demonstrate that LCs have a key role in UVB-induced cutaneous carcinogenesis and suggest that LCs locally stimulate KC proliferation and innate immune cells that provoke tumor outgrowth.

Modulation of cytokine and nitric oxide production by keratinocytes, epithelial cells, and mononuclear phagocytes in a co-culture model of inflammatory acne.

INTRODUCTION: Ultraviolet B (UVB, 290 nm to 320 nm) has been reported to modulate the cytokine-mediated inflammatory process in various inflammatory skin conditions, including production of TNF-α, IL-1α, IL-6, IL-8, and IL-10. We constructed an in vitro model system involving co-culture of different cell types to study the effect of UVB on the inflammatory process using nitric oxide (NO) and tumor necrosis factor (TNF)-α as markers of inflammation. OBJECTIVE: This study was conducted to quantitatively assess the products secreted by human epithelial keratinocytes in the presence and absence of macrophages/monocytes. METHODS: Cells were exposed to UVB radiation (50 mJ to 200 mJ per cm2) or treated with bacterial lipopolysaccharide (LPS) as stimulator of inflammatory response. Nitric oxide (NO) was measured by modified Griess assay and TNF-α was measured by quantitative ELISA. For the co-culture system, SC monocytes were seeded in a 24-well Transwell tissue culture plate whereas irradiated keratinocytes were seeded in the individual baskets subsequently placed on top of the monocyte cultures, and samples of culture supernatants were collected at 1 to 6 days. RESULTS: When primary human epidermal keratinocytes (NHEK) were irradiated with UVB, a dose-dependent stimulation of TNF-α production was observed (33% to 200% increase). TNF-α production was not changed significantly in SC monocytes/NHEK co-culture. In contrast, when macrophages were irradiated with UVB, significant inhibition of NO production (40% suppression, P<0.001) was seen. CONCLUSION: This improved model of cutaneous inflammation could use multiple cells to study their interactions and to offer convenience, reproducibility, and a closer approximation of in vivo conditions.

Mast cell-derived IL-10 suppresses germinal center formation by affecting T follicular helper cell function.

The most prevalent cancer diagnosed in the world is sunlight-induced skin cancer. In addition to being a complete carcinogen, UV radiation, the causative agent of skin cancer, induces immune suppression. Because UV-induced immune suppression is a well-recognized risk factor for skin cancer induction, it is crucial to understand the mechanisms underlying UV-induced immune suppression. Mast cells, which have recently emerged as immune regulatory cells, are particularly important in UV-induced immune suppression. UV exposure does not induce immune suppression in mast cell-deficient mice. We report that UV irradiation blocks germinal center (GC) formation, Ab secretion, and T follicular helper (Tfh) cell function, in part by altering the expression of transcription factors BCL-6 and BLIMP-1. No suppression of GC formation, Tfh cell IL-21 expression, or Ab secretion was observed in UV-irradiated mast cell-deficient (Kit(W-sh/W-sh)) mice. When mast cell-deficient mice were reconstituted with wild type mast cells, immune suppression was restored. Reconstituting the mast cell-deficient mice with bone marrow-derived mast cells from IL-10-deficient mice failed to restore the ability of UV radiation to suppress GC formation. Our findings demonstrate a function for mast cells, suppression of Tfh cell production, GC formation, and Ab production in vivo.

Adenoviral-mediated mda-7 expression suppresses DNA repair capacity and radiosensitizes non-small-cell lung cancer cells.

The melanoma differentiation-associated gene-7 (mda-7) was identified by virtue of its enhanced expression in human melanoma cells induced into terminal differentiation. Enforced expression of mda-7 in human cancer cell lines of diverse origins results in the suppression of growth and induction of apoptosis. We have shown that adenoviral-mediated mda-7 (Ad-mda7) radiosensitizes non-small-cell lung cancer (NSCLC) cells by enhancing the apoptotic pathway. To identify the mechanism of this radiosensitization, we examined the level of proteins involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Western blot analysis indicated that the expression of NHEJ pathway components Ku70, XRCC4, and DNA ligase IV was downregulated in NSCLC cells--A549 with Ad-mda7 treatment. No such change was observed in normal human CCD16 fibroblasts previously shown not to be radiosensitized by Ad-mda7. The biological significance of these changes of expression of proteins critical for repair of radiation-induced DSBs was confirmed via the analysis of DSB rejoining kinetics using pulsed field gel electrophoresis and assessment of host cell reactivation capacity following Ad-mda7 treatment. Based on these results, we hypothesize that Ad-mda7 sensitizes NSCLC cells to ionizing radiation by suppressing the activity of NHEJ, a pathway essential for repair of radiation-induced DSBs.

Can short-term administration of dexamethasone abrogate radiation-induced acute cytokine gene response in lung and modify subsequent molecular responses?

PURPOSE: To investigate the effects of short-term administration of dexamethasone (DEX) on radiation-induced responses in the mouse lung, focusing on expression of pro-inflammatory cytokine and related genes. METHODS AND MATERIALS: At indicated times after thoracic irradiation and/or drug treatment, mRNA expression levels of cytokines (mTNF-alpha, mIL-1 alpha, mIL-1 beta, mIL-2, mIL-3, mIL-4, mIL-5, mIL-6, mIFN-gamma) and related genes in the lungs of C3H/HeN mice were measured by RNase protection assay. RESULTS: Radiation-induced pro-inflammatory cytokine mRNA expression levels in lung peak at 6 h after thoracic irradiation. DEX (5 mg/kg) suppresses both basal cytokine mRNA levels and this early response when given immediately after irradiation. However, by 24 h, in mice treated with DEX alone or DEX plus radiation, there was a strong rebound effect that lasted up to 3 days. Modification of the early radiation-induced response by DEX did not change the second wave of cytokine gene expression in the lung that occurs at 1 to 2 weeks, suggesting that early cytokine gene induction might not determine subsequent molecular events. A single dose of DEX attenuated, but did not completely suppress, increases in cytokine mRNA levels induced by lipopolysaccharide (2.5 mg/kg) treatment, but, unlike with radiation, no significant rebound effect was seen. Five days of dexamethasone treatment in the pneumonitic phase also inhibited pro-inflammatory cytokine gene expression and, again, there was a rebound effect after withdrawal of the drug. CONCLUSIONS: Our findings suggest that short-term use of dexamethasone can temporarily suppress radiation-induced pro-inflammatory cytokine gene expression, but there may be a rebound after drug withdrawal and the drug does little to change the essence and course of the pneumonitic process.

[An experimental in-vitro evaluation of membrane antigen expression and of interleukin production by monocytes exposed to magnetic resonance]. / Valutazione sperimentale in vitro dell'espressione di antigeni di membrana e della produzione di interleuchine da parte di linfomonociti esposti a Risonanza Magnetica.

The interactions of magnetic fields and radiofrequency with biological systems have been described by several authors. However, no definitive conclusions have been drawn yet as to the safety of the magnetic fields and radiofrequencies used in clinical examinations. The immune system is one of the most complex biological systems, in which a network of intracellular signals regulates the immune response. Interleukins are released by an activation process which involves, at least in part, intra- and/or extracellular calcium mobilization. The latter step can be influenced by the in vitro effect of this type of nonionizing radiations produced by an MR system on peripheral blood mononuclear cells. Our results show that the 2-hour exposure to magnetic fields (0.5 T) and radiofrequency caused an increase in the spontaneous release of IL2, IL4, IL10, TNF alpha and INF gamma, while the amount of the same cytokines induced by PHA stimulation was decreased. No differences were observed in the spontaneous or PHA-induced release of IL6 by exposure to magnetic fields (MRI). Furthermore, the expression of the CD 11b molecule was increased at the same time. These results may be useful for us to understand the interactions between magnetic fields and radiofrequencies and the immune system.

Ultraviolet B radiation up-regulates the expression of IL-15 in human skin.

Ultraviolet B (UVB) radiation is a potent modulator of skin-related immune responses, particularly those involving the synthesis and the secretion of cytokines. The discovery of a new T cell mitogen, IL-15, prompted use to investigate its expression in skin and to examine the effects of UVB radiation on such expression. RNA from unirradiated and UVB-irradiated epidermal and dermal sheets derived from human foreskin as well as from unirradiated and UVB-irradiated skin cell populations were assayed for IL-15 expression by semiquantitative RT-PCR. Constitutive levels of IL-15 mRNA were detected in dermal sheets, but not in epidermal sheets. Following UVB treatment, IL-15 mRNA was induced in epidermal sheets and enhanced in dermal sheets. UVB-inducible epidermal expression of IL-15 mRNA was traced to HLA-DR- cells (presumably keratinocytes) and not to HLA-DR+ cells (Langerhans cells). Cultured keratinocytes and dermal fibroblasts displayed basal levels of IL-15 mRNA that were also up-regulated following UVB exposure. Immunoblot analysis revealed secretion of IL-15 protein by keratinocytes that was enhanced following UVB treatment. These results constitute the first report of IL-15 mRNA expression and protein production in human skin. In addition to expanding the known influence of UVB radiation on the capacity of keratinocytes and dermal fibroblasts to express immunomodulatory cytokines, these findings suggest a new mechanism by which UVB can promote Ag-independent T cell responses via elaboration of IL-15.