Site α Is Crucial for Two Routes of IFNγ-Induced MHC Class I Transactivation: The ISRE-Mediated Route and a Novel Pathway Involving CIITA

AbstractThe constitutive and cytokine-induced levels of major histocompatibility (MHC) class I expression are tightly controlled at the transcriptional level. In this study, it is shown that the cis-acting regulatory element site α of the MHC class I promoter is essential for the IFNγ-induced transactivation of MHC class I gene expression through the ISRE. Moreover, it was discovered that the class II transactivator (CIITA), which is itself under the control of the IFNγ induction pathway, strongly transactivates MHC class I gene expression and exerts its activity through site α. Therefore, site α is a crucial regulatory element, mediating the classic route of IFNγ induction via the ISRE as well as a novel route of MHC class I transactivation involving CIITA

Humanized mouse model of skin inflammation is characterized by disturbed keratinocyte differentiation and influx of IL-17A producing T cells

Contains fulltext : 108173.pdf (publisher's version ) (Open Access)Humanized mouse models offer a challenging possibility to study human cell function in vivo. In the huPBL-SCID-huSkin allograft model human skin is transplanted onto immunodeficient mice and allowed to heal. Thereafter allogeneic human peripheral blood mononuclear cells are infused intra peritoneally to induce T cell mediated inflammation and microvessel destruction of the human skin. This model has great potential for in vivo study of human immune cells in (skin) inflammatory processes and for preclinical screening of systemically administered immunomodulating agents. Here we studied the inflammatory skin response of human keratinocytes and human T cells and the concomitant systemic human T cell response.As new findings in the inflamed human skin of the huPBL-SCID-huSkin model we here identified: 1. Parameters of dermal pathology that enable precise quantification of the local skin inflammatory response exemplified by acanthosis, increased expression of human beta-defensin-2, Elafin, K16, Ki67 and reduced expression of K10 by microscopy and immunohistochemistry. 2. Induction of human cytokines and chemokines using quantitative real-time PCR. 3. Influx of inflammation associated IL-17A-producing human CD4+ and CD8+ T cells as well as immunoregulatory CD4+Foxp3+ cells using immunohistochemistry and -fluorescence, suggesting that active immune regulation is taking place locally in the inflamed skin. 4. Systemic responses that revealed activated and proliferating human CD4+ and CD8+ T cells that acquired homing marker expression of CD62L and CLA. Finally, we demonstrated the value of the newly identified parameters by showing significant changes upon systemic treatment with the T cell inhibitory agents cyclosporine-A and rapamycin. In summary, here we equipped the huPBL-SCID-huSkin humanized mouse model with relevant tools not only to quantify the inflammatory dermal response, but also to monitor the peripheral immune status. This combined approach will gain our understanding of the dermal immunopathology in humans and benefit the development of novel therapeutics for controlling inflammatory skin diseases

TNF-α promoter polymorphisms, production and susceptibility to multiple sclerosis in different groups of patients

TNF-α production in whole blood cultures upon stimulation with LPS was determined in 179 individuals from 61 families in order to characterise the magnitude of inherited differences in TNF-α production. The three families characterised by highest TNF production showed 7.1 ± 0.3 ng TNF/ml upon culture with 10 ng LPS and 10.2 ± 0.2 ng TNF/ml upon culture with 1000 ng LPS, in contrast to the three families characterised by the lowest TNF production that showed a production of 1.6 ± 0.1 ng TNF upon culture with 10 ng and 2.5 ± 0.2 ng/ml upon culture with 1000 ng LPS/ml. This difference could not be attributed to the promoter polymorphisms -308 G to A, -238 G to A or -376 G to A, although the -238 GA donors produced 2.1 ± 0.9 ng TNF upon culture with 10 ng endotoxin compared to 3.2 ± 2.2 ng TNF for the -238 GG donors. In line with these results the frequency of the -238 GG genotype was increased in hospitalized MS patients in a nursing home (100% 238GG, n = 57) compared to MS patients in an outpatient's clinic (94% 238GG, n = 98) or Dutch controls (90% 238GG, n = 180). These results suggest that the -238 GG genotype is differently distributed in hospitalized MS patients in a nursing home

Acanthosis and aberrant epidermal marker expression of hBD2, Elafin, K10 and K16 in the inflamed human skin of the huPBL-SCID-hu Skin allograft model.

<p><b>A.</b> Histology (H&E staining) of human skin grafts from SCID beige mice 21 days after infusion (i.p.) of huPBMC. Representative photographs are shown after PBS (left photograph) and huPBMC infusion (right photograph). Photograph shows increased epidermal thickness (acanthosis) and elongated fingerlike epidermal projections into the dermis (rete ridges) and large lymphocyte infiltration after huPBMC infusion. Abnormal presence of nuclei in the stratum corneum (parakeratosis) and infiltration of lymphocytes in the epidermis (exocytosis) (bottom-right photograph). 20×, 20×, 40× magnifications respectively. <b>B.</b> Quantitative microscopic histological analysis of the epidermal thickness (µm) of human skin grafts following infusion of PBS and huPBMC. Mean±SEM are shown for n = 3 and 6 upon PBS and huPBMC infusion resp. <b>C.</b> Immunohistochemistry of K10, K16, hBD2 and Elafin (brown) in human skin grafts from SCID beige mice 21 days after infusion (i.p.) of PBS (top panels) or huPBMC (lower panels). Photograpsh show representative examples, summarized data are given in the figures. Mean±SEM percentages of the area positive for the indicated markers is shown for n = 3 and 5–8 upon PBS and huPBMC infusion resp. 10× magnification. <b>D.</b> Ki-67 expression (brown) in the stratum basale of the epidermis. The insert shows a higher magnification. A representative example of n = 12 is shown. 20× magnification. Graphs show summarized data of Ki67+ cells/mm length of basement membrane after PBS or huPBMC infusion in human skin biopsies (mean±SEM, of n = 4 and 6, resp.).</p

Infiltration of human IL-17A-producing T cells and CD4+ Foxp3-expressing T cells in the inflamed human skin in the SCID/skin allograft mouse model.

<p><b>A.</b> Immunohistochemistry of human CD4 (brown, top) and CD8 (brown, bottom) expression in human skin grafts from SCID beige mice 21 days after infusion or huPBMC. Photographs show representative examples. 20× magnification. <b>B.</b> Summarized data of figure A. showing mean±SEM CD4 (top) or CD8 (bottom) positive cells/mm² of n = 4 and 10 upon PBS and huPBMC infusion resp in the epidermis (white bars) and dermis (black bars). <b>C.</b> Immunohistochemistry of human IL-17A expression in human skin grafts from SCID beige mice 21 days after infusion of PBS (left) or huPBMC (right). Photographs show representative examples of n = 6 (huPBMCs) n = 3 (controls). 20× magnification. Graph shows summarized data of IL-17A positive cells/mm² following PBS or huPBMC infusion in the human skin biopsies (mean±SEM, of n = 4 and 10). <b>D.</b> Immunohistochemistry of coexpression of human CD4 (blue) and IL-17A (red, top) and CD8 (blue) and IL-17A (red, bottom) in human skin grafts from SCID beige mice 21 days after infusion of huPBMC (20× magnification). Inserts show a higher magnification (40×) of single CD4/CD8 and IL-17A staining and CD4/IL-17A or CD8/IL-17A co-staining. . Photographs show representative examples of n = 6. <b>E.</b> Immunohistochemistry of IL-17A (red) in human mastcell tryptase (brown) and granulocyte elastase (brown) in human skin biopsies from SCID beige mice 21 days after infusion of PBS (left) or huPBMC (right). Photographs show representative examples of n = 6 (huPBMCs) n = 3 (controls). 20× magnification. <b>F.</b> Immunohistochemistry of co-expression of human Foxp3 (brown) and CD4 (blue) in human skin grafts from SCID beige mice 21 days after infusion of PBS (left) or huPBMC (right) (40× magnification). Inserts show a higher magnification (63×) of single Foxp3 and CD4+ staining and Foxp3/CD4 co-staining. Photographs show representative examples of n = 6 (huPBMCs) n = 3 (controls).</p

Cyclosporin-A and rapamycin treatment restores aberrant human epidermal differentiation marker expression and prevents infiltration of inflammation associated IL-17A producing human T cells of the human skin in the huPBL-SCID/skin allograft mouse model.

<p><b>A.</b> Macroscopic (top panel) and histological (H&E) (lower panel) appearance of human skin graft from SCID beige mice 21 days after infusion of huPBMC with no treatment (left panel) and treatment with CsA and Rapa (right panel). <b>B.</b> Quantitative microscopic histological analysis of the epidermal thickness (µm) of skin grafts following infusion of huPBMC with or without CsA and Rapa. Mean±SEM are shown for n = 3 and 5 upon PBS and huPBMC infusion resp. 20× magnification. <b>C.</b> Immunohistochemistry of K10, K16, hBD2 and Elafin in human skin grafts from SCID beige mice 21 days after infusion (i.p.) of huPBMC without (top panel) and with CsA and Rapa treatment (lower panel). Representative examples are show. 10× magnification. <b>D.</b> Summarized data represented in C are given in the figures. Mean±SEM percentages of the area positive for the indicated markers is shown for n = 3 and 4–6 upon no treatment and treatment with CsA and Rapa, resp. <b>E.</b> Immunohistochemistry of CD4 (top panel) and CD8 (lower panel) in human skin grafts from SCID beige mice 21 days after infusion (i.p.) of huPBMC without (left panel) and with CsA and Rapa treatment (right panel). Representative examples are show. 20× magnification. <b>F.</b> Summarized data represented in E. are given in the figures. Mean±SEM percentages of CD4 (top) and CD8 (bottom) positive cells is shown for n = 3 and 4–5 upon no treatment and treatment with CsA and Rapa, resp. <b>G.</b> Immunohistochemistry of IL-17A (brown) (representative examples of n = 5 are shown) 10× magnification. Graph shows summarized data of IL-17A positive cells/mm² present in human skin biopsies following infusion of huPBMC in the absence or presence of Rap/CsA resp. (mean±SEM, of n = 3 and 4–5).</p

Induction of human chemokines and cytokines and influx of human T cells in the dermis and in the epidermis of huPBL-SCID-hu Skin allograft model.

<p><b>A.</b> Images show representative panoramic overviews (H&E staining) of human skin grafts from SCID beige mice 21 days after infusion (i.p.) of PBS (top) or huPBMC (below). Note that human epidermis is thicker than the mouse epidermis (present in the left edge of the specimen in the top image and in both edges in the bottom image) and in contrast with human skin, mouse skin has closely spaced hair folicules troughout the epidermis. Images were composed using PTGui software (New House Internet Services B.V.; <a href="http://www.ptgui.com/" target="_blank">http://www.ptgui.com/</a>). <b>B.</b> Immunohistochemistry of human CD3+ (brown) T cell infiltration in the human dermis and epidermis, 21 days after infusion of huPBMC. Representative example of n = 12 are shown. 20× magnification. <b>C.</b> Gene expression analysis using quantitative real-timePCR of human cytokine and chemokine transcripts in the human skin 21 days after infusion of huPBMC. Figure shows fold increase in cytokine and chemokine mRNA expression levels after huPBMC infusion as compared to PBS infusion (n = 5 and 3; huPBMC and PBS resp.).</p