Roswitha Sgonc

Avian models with spontaneous autoimmune diseases.

Autoimmune diseases in human patients only become clinically manifest when the disease process has developed to a stage where functional compensation by the afflicted organ or system is not possible anymore. In order to understand the initial etiologic and pathogenic events that are generally not yet accessible in humans, appropriate animal models are required. In this respect, spontaneously developing models--albeit rare--reflect the situation in humans much more closely than experimentally induced models, including knockout and transgenic mice. The present chapter describes three spontaneous chicken models for human autoimmune diseases, the Obese strain (OS) with a Hashimoto-like autoimmune thyroiditis, the University of California at Davis lines 200 and 206 (UCD-200 and -206) with a scleroderma-like disease, and the amelanotic Smyth line with a vitiligo-like syndrome (SLV). Special emphasis is given to the new opportunities to unravel the genetic basis of these diseases in view of the recently completed sequencing of the chicken genome.

Pro- and anti-fibrotic effects of TGF-β in scleroderma

The University of California at Davis 200 and 206 (UCD-200/206) lines of chickens have proven to be the animal model that best reflects the situation in human SSc. We have demonstrated a misbalance of pro-fibrotic (TGF-beta1) and anti-fibrotic (TGF-beta2 and -beta3) TGF-beta isoforms as a possible cause for fibrotic alterations in this model. This opens new avenues for diagnosis and therapy for this still intractable condition.

Replenishment of the B cell compartment after doxorubicin-induced hematopoietic toxicity is facilitated by STAT1

STAT1 serves as an important regulator in the response to pathogens, oncogenic transformation, and genotoxic insults. It exerts these effects by shaping the innate and adaptive immune response and by participating in genotoxic stress pathways, leading to apoptosis and inhibition of cell proliferation. We have investigated the role of STAT1 in hematopoietic toxicity induced by doxorubicin in STAT1‐proficient and ‐deficient mice. Whereas the early genotoxic effect of doxorubicin did not depend on STAT1, expression of STAT1 was required for efficient B lymphocyte repopulation in the recovery phase. We found a lower abundance of lymphocyte precursors in the BM of STAT1‐deficient animals, which was particularly evident after doxorubicin‐induced hematopoietic toxicity. In accordance, colony‐forming assays with STAT1‐deficient BM cells revealed a decreased number of pre‐B colonies. Differentiation from the pro‐B to the pre‐B stage was not affected, as demonstrated by unaltered differentiation of purified B cell precursors from BM in the presence of IL‐7. With the exception of Sca‐1, expression of genes implicated in early lymphocyte development in pro‐B cells did not depend on STAT1. Our findings indicate a specific requirement for STAT1 in lymphoid development before differentiation to pre‐B cells, which becomes particularly apparent in the recovery phase from doxorubicin‐induced hematopoietic toxicity.

The BH3-only protein Bad is dispensable for TNF-mediated cell death

Tumor necrosis factor (TNF) is a key signaling molecule orchestrating immune and inflammatory responses and possesses the capacity to trigger apoptotic as well as necroptotic cell death. Apoptotic cell death elicited by TNF has been demonstrated to engage pro-apoptotic Bcl-2 family proteins, most prominently the BH3-only protein Bid, a key substrate of caspase-8, the key effector protease downstream of TNF receptor I. Most recently, the BH3 domain-containing protein Bad (Bcl-2-antagonist of cell death) has been shown to be rate limiting for TNF-mediated cell death, suggesting possible synergy with Bid, but genetic analyses presented here demonstrate that it is dispensable for this process.

Mapping QTL affecting a systemic sclerosis-like disorder in a cross between UCD-200 and red jungle fowl chickens

Systemic sclerosis (SSc) or scleroderma is a rare, autoimmune, multi-factorial disease characterized by early microvascular alterations, inflammation, and fibrosis. Chickens from the UCD-200 line develop a hereditary SSc-like disease, showing all the hallmarks of the human disorder, which makes this line a promising model to study genetic factors underlying the disease. A backcross was generated between UCD-200 chickens and its wild ancestor - the red jungle fowl and a genome-scan was performed to identify loci affecting early (21 days of age) and late (175 days of age) ischemic lesions of the comb. A significant difference in frequency of disease was observed between sexes in the BC population, where the homogametic males were more affected than females, and there was evidence for a protective W chromosome effect. Three suggestive disease predisposing loci were mapped to chromosomes 2, 12 and 14. Three orthologues of genes implicated in human SSc are located in the QTL region on chromosome 2, TGFRB1, EXOC2-IRF4 and COL1A2, as well as CCR8, which is more generally related to immune function. IGFBP3 is also located within the QTL on chromosome 2 and earlier studies have showed increased IGFBP3 serum levels in SSc patients. To our knowledge, this study is the first to reveal a potential genetic association between IGFBP3 and SSc. Another gene with an immunological function, SOCS1, is located in the QTL region on chromosome 14. These results illustrate the usefulness of the UCD-200 chicken as a model of human SSc and motivate further in-depth functional studies of the implicated candidate genes.

Efficient therapy of ischaemic lesions with VEGF121-fibrin in an animal model of systemic sclerosis.

Background In systemic sclerosis (SSc), chronic and uncontrolled overexpression of vascular endothelial growth factor (VEGF) results in chaotic vessels, and intractable fingertip ulcers. Vice versa, VEGF is a potent mediator of angiogenesis if temporally and spatially controlled. We have addressed this therapeutic dilemma in SSc by a novel approach using a VEGF121 variant that covalently binds to fibrin and gets released on demand by cellular enzymatic activity. Using University of California at Davis (UCD)-206 chickens, we tested the hypothesis that cell-demanded release of fibrin-bound VEGF121 leads to the formation of stable blood vessels, and clinical improvement of ischaemic lesions. Methods Ninety-one early and late ischaemic comb and neck skin lesions of UCD-206 chickens were treated locally with VEGF121-fibrin, fibrin alone, or left untreated. After 1u2005week of treatment the clinical outcome was assessed. Angiogenesis was studied by immunofluorescence staining of vascular markers quantitatively analysed using TissueQuest. Results Overall, 79.3% of the lesions treated with VEGF121-fibrin showed clinical improvement, whereas 71.0% of fibrin treated controls, and 93.1% of untreated lesions deteriorated. This was accompanied by significantly increased growth of stable microvessels, upregulation of the proangiogenic VEGFR-2 and its regulator TAL-1, and increase of endogenous endothelial VEGF expression. Conclusions Our findings in the avian model of SSc suggest that cell-demanded release of VEGF121 from fibrin matrix induces controlled angiogenesis by differential regulation of VEGFR-1 and VEGFR-2 expression, shifting the balance towards the proangiogenic VEGFR-2. The study shows the potential of covalently conjugated VEGF-fibrin matrices for the therapy of ischaemic lesions such as fingertip ulcers.

DNA-binding of the Tet-transactivator curtails antigen-induced lymphocyte activation in mice

The Tet-On/Off system for conditional transgene expression constitutes state-of-the-art technology to study gene function by facilitating inducible expression in a timed and reversible manner. Several studies documented the suitability and versatility of this system to trace lymphocyte fate and to conditionally express oncogenes or silence tumour suppressor genes in vivo. Here, we show that expression of the tetracycline/doxycycline-controlled Tet-transactivator, while tolerated well during development and in immunologically unchallenged animals, impairs the expansion of antigen-stimulated T and B cells and thereby curtails adaptive immune responses in vivo. Transactivator-mediated cytotoxicity depends on DNA binding, but can be overcome by BCL2 overexpression, suggesting that apoptosis induction upon lymphocyte activation limits cellular and humoral immune responses. Our findings suggest a possible system-intrinsic biological bias of the Tet-On/Off system in vivo that will favour the outgrowth of apoptosis resistant clones, thus possibly confounding data published using such systems.Tet-transactivators are used for direct regulation of gene expression, RNA interference and for CRISPR/Cas9-based systems. Here the authors show that DNA-bound Tet-transactivators can induce cell death in antigen-activated lymphocytes in vivo, putting into question the use of, and in vivo data generated with, these molecular tools.

Correction to: The BH3-only protein Bad is dispensable for TNF-mediated cell death

Since publication of this article, the authors wished to draw attention to an error in the materials section as a result of which they have been mis-cited (https://www.nature.com/articles/s41422-018-0041-7). The dose of TNF given was not in fact 15u2009mg/kg body weight (as stated in the “mouse work” section), but 15u2009μg/kg body weight.

AB0206 Altered Expression of Angiopoietins and Tie-2 in Ischemic Lesions of UCD-206 Chickens, an Animal Model for Systemic Sclerosis

Background Systemic sclerosis (SSc) is an autoimmune connective tissue disease affecting skin and internal organs. It is characterized by vascular damage, inflammation, and fibrosis. Microvascular injury and insufficient angiogenesis lead to tissue ischemia and clinical manifestations such as fingertip ulcers. UCD-206 chickens are the only animal model showing all hallmarks of the human disease, including microvascular damage and insufficient angiogenesis. The major inducer of angiogenesis is vascular endothelial growth factor (VEGF). Uncontrolled expression of VEGF and its receptors VEGFR-1 and VEGFR-2 has been shown in both, human and avian SSc. Another set of molecules that critically regulate angiogenesis are the endothelial cell-specific receptor tyrosine kinase 2 (Tie-2) and its ligands angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2). Although abnormal plasma levels of soluble sTie-2, sAng-1, and sAng-2 have been reported in SSc patients, expression in lesional tissues has not been examined. Objectives To study the expression of Tie-2, Ang-1 and Ang-2 in lesional and non-lesional skin of UCD-206 chickens. Methods The expression of Tie-2, Ang-1 and Ang-2 was studied by indirect immunofluorescence tests on frozen tissue sections from lesional and non-lesional neck skins from 5 weeks old UCD-206 chickens. Age matched H.B15 chickens were used as healthy controls. Samples were double stained with monoclonal anti-Tie-2 antibody and the endothelial cell marker anti-von Willebrand factor (vWF), monoclonal anti-Ang-1 antibody and anti-alpha smooth muscle actin antibody, as a marker for mural cells, or with monoclonal anti-Ang-2 antibody and anti-vWF. The tissue cytometry software TissueQuest® was used for quantitative analyses. Statistical significance was calculated using the Mann-Whitney-U test. Results The number of Tie-2 expressing endothelial cells was significantly increased in lesional skin compared to non-lesional UCD-206 skin and healthy controls. Expression of Ang-2 was significantly decreased in lesional compared to non-lesional UCD-206 skin and to healthy H.B15 skin. Whereas no significant difference was seen in the numbers of Ang-1 expressing mural cells, other Ang-1 expressing cells were reduced in lesional skin compared to non-lesional and healthy controls. The altered endothelial expression of Ang-2 and Tie-2 resulted in significantly decreased Ang-2:Tie-2 ratios in lesional (median 0.9, IQR 0.3 – 1.8) compared to non-lesional UCD-206 skin (median 6.9, IQR 3.4 – 14.2, p≤0.0005) and healthy controls. (median 11.2, IQR 2.4 – 25.2, p≤0.0005). The Ang-2:Ang-1 ratio was also significantly diminished in lesional (median 0.4, IQR 0.1 – 0.5) compared to non-lesional UCD-206 skin (median 0.7, IQR 0.4 – 1.3, p≤0.01) and healthy controls. (median 1.0, IQR 0.6 – 1.6, p≤0.002). Conclusions Altered expression of Ang-1, Ang-2 and Tie-2 might contribute to the vasculopathy in SSc. Acknowledgements This work was funded by the Austrian Science Fund (FWF): P23230-B13. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2338

OP0300 Effect of VEGF121-Fibrin on the Expression of VEGF Receptors in Ischemic Lesions of UCD-206 Chickens, an Animal Model for Systemic Sclerosis

Background Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vascular alterations, perivascular mononuclear cell infiltration, and fibrosis of skin and viscera. Microvascular injury, disturbed vascular repair and insufficient angiogenesis result in an overall reduced capillary density, and clinical manifestations such as fingertip ulcers. Expression of vascular endothelial cell growth factor (VEGF), the most potent angiogenic factor, and its receptors VEGFR-1 and VEGFR-2 is highly upregulated in SSc skin, but seems to be uncontrolled and chronic. However, a very high VEGF expression is associated with the lack of fingertip ulcers. Therefore, local administration of VEGF in a form that allows controlled release might be a promising therapeutic approach. Objectives Based on a proof of concept study in UCD-206 chickens, an animal model showing all hallmarks of the human disease, where we showed that cell demanded release of locally applied exogenous matrix-bound VEGF121-fibrin leads to the formation of stable blood vessels, and clinical improvement of ischemic lesions, we analysed the effect of this therapeutic approach on VEGFR-1 and VEGFR-2 expression. Methods Early and late ischemic comb and neck skin lesions of UCD-206 chickens were treated locally with VEGF121-fibrin or as a placebo control with fibrin alone. After 7 days the clinical outcome was assessed, and skin biopsies were taken for further analyses. To study the expression of VEGFR-1 and VEGFR-2 on endothelial cells, frozen tissue sections were double stained with the respective VEGF-receptor specific antibody and anti-van Willebrand factor (vWF) antibody by indirect immunofluorescence tests. The numbers of VEGFR-1 positive and VEGFR-2 positive endothelial cells were quantified using Tissuequest. Statistical significance was calculated using the Mann-Whitney-U test. Results Seven days after treatment with VEGF121-fibrin approximately 80% of the lesions showed clear clinical improvement, whereas 82% of placebo controls and 97% of untreated lesions had deteriorated. Angiogenesis was significantly increased after treatment with VEGF121-fibrin compared to controls. VEGF121-fibrin treatment increased the expression of the pro-angiogenic receptor VEGFR-2, and strongly reduced the endothelial expression of the modulatory or anti-angiogenic receptor VEGFR-1. Conclusions In the avian SSc model, the cell demanded release of VEGF121 from fibrin seems to result in controlled angiogenesis with formation of morphological normal blood vessels. The released VEGF differentially regulates the expression of VEGFR-1 and VEGFR-2 shifting the balance towards the pro-angiogenic VEGFR-2, thus increasing the angiogenic response. Long term effects of VEGF121-fibrin on vessel stability, the expression of endogenous VEGF, and its receptors will be investigated in a follow up study. Disclosure of Interest None Declared