Organ transplantation and skin--principles and concepts

Solid organ transplantation influences the biology of the skin profoundly. In the wake of transplantation, inflammatory, infectious and neoplastic disorders arise, often with atypical clinical presentation. Inflammatory disorders mainly relate to pathogen-driven conditions such as seborrheic dermatitis and pityrosporum folliculitis and to drug reactions. Infectious disorders are dominated by viral infections of human papilloma virus and by infections and reactivations of herpes family members. Neoplastic disorders are greatly increased with squamous cell carcinoma of the skin as most relevant clinical problem which is increased 65- to 100-fold following transplantation. This dramatic increase in cutaneous carcinogenesis results from the isolated effect of ultraviolet light on the skin with immunosuppression and DNA damage and of immunosuppressants which drive skin cancer formation by properties unrelated to immunosuppression and from the combined effect of UV light and immunosuppressive drugs on immunomodulation which results in impaired antitumor response as well as chronic tumorigenic inflammation

Alien Registration- Gaoosselin, Mary O. (Augusta, Kennebec County)

https://digitalmaine.com/alien_docs/18929/thumbnail.jp

TLR4 as a negative regulator of keratinocyte proliferation

TLR4 is an innate immune receptor with expression in human skin, keratinocytes as well as squamous cell carcinoma (SCC) of the skin. In the present study we investigate the role of TLR4 as a negative regulator of keratinocyte proliferation. We present here that the expression of TLR4 increased with the differentiation of cultured keratinocytes in a passage-dependent manner or under calcium-rich conditions. Moreover, the down-regulation of TLR4 by specific knockdown increased the proliferation of HaCaT keratinocytes in vitro. In addition, subcutaneously injected HaCaT keratinocytes with shTLR4 formed growing tumors in nude mice. In contrast, we observed lower proliferation and increased migration in vitro of the SCC13 cell line stably overexpressing TLR4 in comparison to SCC13 TLR4 negative cells. In vivo, SCC13 TLR4-overexpressing tumors showed delayed growth in comparison to TLR4 negative tumors. The overexpression of TLR4 in SCC13 tumor cells was followed by phosphorylation of ERK1/2 and JNK and increased expression of ATF3. In gene expression arrays, the overexpression of TLR4 in tumor cells correlated with gene expression of ATF-3, IL-6, CDH13, CXCL-1 and TFPI. In summary, TLR4 negatively regulates the proliferation of keratinocytes and its overexpression reduces tumor growth of SCC cells

Ingenol mebutate signals via PKC/MEK/ERK in keratinocytes and induces interleukin decoy receptors IL1R2 and IL13RA2

Squamous cell carcinoma (SCC) is the second most common human skin cancer and the second leading cause of skin cancer-related death. Recently, a new compound, ingenol mebutate, was approved for treatment of actinic keratosis, a precursor of SCC. As the mechanism of action is poorly understood, we have further investigated the mechanism of ingenol mebutate-induced cell death. We elucidate direct effects of ingenol mebutate on primary keratinocytes, patient-derived SCC cells, and a SCC cell line. Transcriptional profiling followed by pathway analysis was performed on ingenol mebutate-treated primary keratinocytes and patient-derived SCC cells to find key mediators and identify the mechanism of action. Activation of the resulting pathways was confirmed in cells and human skin explants and supported by a phosphorylation screen of treated primary cells. The necessity of these pathways was demonstrated by inhibition of certain pathway components. Ingenol mebutate inhibited viability and proliferation of all keratinocyte-derived cells in a biphasic manner. Transcriptional profiling identified the involvement of PKC/MEK/ERK signaling in the mechanism of action and inhibition of this signaling pathway rescued ingenol mebutate-induced cell death after treatment with 100 nmol/L ingenol mebutate, the optimal concentration for the first peak of response. We found the interleukin decoy receptors IL1R2 and IL13RA2 induced by ingenol mebutate in a PKC/MEK/ERK-dependent manner. Furthermore, siRNA knockdown of IL1R2 and IL13RA2 partially rescued ingenol mebutate-treated cells. In conclusion, we have shown that ingenol mebutate-induced cell death is mediated through the PKCδ/MEK/ERK pathway, and we have functionally linked the downstream induction of IL1R2 and IL13RA2 expression to the reduced viability of ingenol mebutate-treated cells. Mol Cancer Ther; 14(9); 2132-42. ©2015 AACR

S100A8/A9 Stimulates Keratinocyte Proliferation in the Development of Squamous Cell Carcinoma of the Skin via the Receptor for Advanced Glycation-End Products

<div><p>Squamous cell carcinoma (SCC) is the most common neoplasm in organ transplant recipients (OTR) on long-term immunosuppression and occurs 60- to 100-fold more frequently than in the general population. Here, we present the receptor for advanced glycation end products (RAGE) and S100A8/A9 as important factors driving normal and tumor keratinocyte proliferation. RAGE and S100A8/A9 were transcriptionally upregulated in SCC compared to normal epidermis, as well as in OTR compared to immunocompetent patients (IC) with SCC. The proliferation of normal and SCC keratinocytes was induced by exposure to exogenous S100A8/A9 which in turn was abolished by blocking of RAGE. The migratory activities of normal and SCC keratinocytes were also increased upon exposure to S100A8/A9. We demonstrated that exogenous S100A8/A9 induces phosphorylation of p38 and SAPK/JNK followed by activation of ERK1/2. We hypothesize that RAGE and S100A8/A9 contribute to the development of human SCC by modulating keratinocyte growth and migration. These processes do not seem to be impaired by profound drug-mediated immunosuppression in OTR.</p></div

The receptor RAGE critically mediates the cellular response to S100A8/A9.

<p>a: Direct blockade by a specific RAGE blocking antibody reduces cellular proliferation. RAGE dependent proliferation was analyzed in normal primary, AK and SCC cells. Cells were incubated for 1 hour with a blocking anti-RAGE antibody (80μg/ml, as recommended by manufacturer) followed by S100A8/A9 stimulation for additional 24 hours. The differences in the proliferation after the blockade were assessed by BrdU incorporation (1 way Anova, Bonferroni`s Multiple test, **p<0.05, ***p<0.05). b: Knockdown of RAGE using shRNA reduces cellular proliferation. The knockdown studies were performed using specific lentiviral shRNA against RAGE. Primary keratinocytes were infected by shRAGE and sh control viral particles. Selection of positive clones was performed by puromycine selection. Cells were grown to optimal confluence and were stimulated with 10ng/ml S100A8/A9.Cells with RAGE knockdown showed a delay in proliferation in comparison to control as assessed by BrdU (70%) (1 way Anova, Bonferroni`s Multiple test p****<0.0001) and microscope images. Exogenous S100A8/A9 did not induce proliferation of shRAGE keratinocytes, but only in the control. c: Blocking TLR4 using specific blocking antibody (HTA125) does not impair cellular proliferation. AK cells were treated with a specific blocking TLR4 antibody (HTA125). AK cells were grown for 24 hours in the presence of HTA125 antibody (1μg/ml) and S100A8/A9 (10ng/ml). For detection of the cellular proliferation rate BrdU proliferation assay was performed.</p

Endogenous S100A8/A9 is involved in cellular proliferation.

<p>a: Spontaneous secretion of S100A8/A9 from normal and SCC-derived keratinocytes. Normal and SCC-derived keratinocytes were grown in 96 well plates for 24 hours. Afterwards, supernatant was collected and preceded for the assessment of secreted S100A8/A9 using specific ELISA for S100A8/A9. b: Blockade of RAGE using anti RAGE blocking antibody reduces spontaneous proliferation of keratinocytes. Normal and SCC-derived keratinocytes were incubated with a blocking anti-RAGE antibody (8ug/100μl) for 24 hours. A decrease of the proliferation rate was detected (20–30%) based on the BrdU incorporation (t-test *p = 0.002). All the results are presented as percentage deviation from corresponding control and represent the mean +/- SD of duplicate values.</p

Exogenous S100A8/A9 induces migration of normal and SCC-derived primary keratinocytes.

<p>a, b: Cells were treated with different concentrations of purified S100A8/A9 (0.01–1 μg/ml) for 15h. The assessment of the migratory potential was analyzed by scratch assay where the number of migrated cells was analyzed. The migration of both normal and SCC-derived keratinocytes was increased significantly (between 60–100% depending on cell type) when 0.01 and 0.1μg/ml of S100A8/A9 were used (t-test, PK, *p = 0.003, *p = 0.004; SCC, *p = 0.0014,*p = 0.0015).</p

Growth of shTLR4 cells in nude mice.

<p>(A) Knockdown of TLR4 induces growth of HaCaT cells in nude mice (injected 4X10³ cells/mouse (mice, n = 6/group); p = 0.0002, t-test). The graphic represents the tumor growth (tumor volume) at different time intervals post injection. (B) Expression of TLR4 in the control and sh TLR4 tumors on end day. The differential TLR4 expression was evaluated on transcriptional level by qPCR using specific anti-human TLR4 primers. The tumor RNA and cDNA synthesis were performed using TRIzol reagent and RT respectively.</p

TLR4 expression increases with the differentiation of normal and SCC keratinocytes in vitro.

<p>(A) Primary keratinocytes were grown in duplicates (n = 2) till full confluency at 96h and the expression of TLR4 and keratinocyte differentiation markers (involucrin and filaggrin) was compared that of low passage (growing) keratinocytes by western blot using specific antibodies. The results were reproducible in two independent experiments. (B) Primary and SCC-derived keratinocytes were grown in duplicates (n = 2) in the presence of Ca²⁺ for different time intervals: 24h, 48h and 96 h. The group “no calcium” was used as positive control for differentiation at 96 hours, the time point of maximal differentiation induced by cell-cell contact. The expression of TLR4 and involucrin was analyzed by western blot using specific antibodies. The results were reproducible in two independent experiments.</p