Conference Scene: Lessons from Animal Models of Autoimmune Diseases: From Mechanisms to Applications

Abstract

Autoimmune diseases are a group of maladies in which the patient‘s immune homeostasis becomes so deregulated that it mounts a destructive attack against the hosts tissues. Such diseases are characterized by an activation of autoreactive T and B cells and are associated, in some cases, with the production of pathogenic antibodies against self-molecules, culminating in inflammation and tissue damage. Target tissues can be from immune-vulnerable and immune-privileged sites. In view of the complex nature of autoimmune diseases, it is not surprising that they have long baffled immunologists, physicians and basic biomedical scientists who are struggling to combine known immunoinflammatory mechanisms into a unified general theory. The present seminar, organized by Euroscion, hosted a group of national and international scientists, affiliated to both academic and industrial research, to discuss state-of-the-art animal models for investigating pathomechanisms of autoimmune diseases, novel laboratory-based diagnostics and novel therapeutic prospects. The timely event on this important topic covered significant features of the basic pathomechanisms of autoimmune disease per se, the development of diagnostic and prognostic tests using functional biomarkers for monitoring patients and the development of targeted therapies. The absence of several prescheduled speakers allowed younger scientists, Stefan Kürten, Liliane Fossati-Jimack and Allan Holmes to shine. We are grateful for their participation. This meeting report describes key points and themes arising from this conference.

Professor Richard Williams (Imperial College London, UK) got the meeting started by announcing that at least three speakers could not be present. Among the absentees was Stephen Thompson (King‘s College London, UK) who was to present his work on the importance of stress proteins in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). This was a disappointment as it is now well documented that stress proteins not only stimulate the host‘s immune system, by acting as mimic autoantigens (which are upregulated in various inflammatory conditions) but also as anti-inflammatory mediators, targeting regulatory T cells [1].

The meeting was dominated by presentations detailing the use of animal models in order to identify and study different targets associated with autoimmune diseases. An exception was vitiligo, for which Pranab K Das of the University of Amsterdam (The Netherlands) presented data obtained primarily using patients. However, although the uses of animal models was the main theme of the meeting, all speakers carefully demonstrated how the fruits of their studies could be translated to identifying targets and possible treatments for the different autoimmune diseases modeled.

The main theme of all presentations revolved round the pivotal role of T-cell subsets (Th1, Th2 and Th17), their associated cytokines (e.g., IL-17/23, IL-20, IL-12, INF-γ, TNF-α and IL-1) and their interactions with antigen-presenting cells (APCs).

As Thompson was absent, Williams opened the meeting by presenting his own groups‘ work on RA and the meeting got underway in the right scientific spirit.

Rheumatoid arthritis & models

Williams concentrated on the interplay between pro- and anti-inflammatory mediators (cytokines and their receptors, chemokines) in the pathogenesis of autoimmune disease. He provided a valuable and extensive summary of different RA mouse models, including immunized mice models (treated with either adjuvants, cartilage antigens or methylated bovine serum albumin) and spontaneous arthritis models such as the huTNF-α transgenic mice, K/BxN mice (a cross between KRN/CB57BL/6-TCR transgenic mice) and SKG mice.

The presentation provided a very clear insight into why the interplay between APCs, T-cell subsets and B cells on the one hand, and TNF-α and IL-17 on the other hand, dominates ideas on the development of arthritis [2]. The rationale for developing treatments for arthritis aimed at inhibiting the actions of TNF-α was compelling and it is not surprising that a number of anti-TNF-α products have been shown to be effective in patients. However, approximately 30% of patients do not respond optimally to anti-TNF-α treatments and it would help if we could better understand the mode of action of the drugs, for example, whether the drugs inhibit both Th1 and Th17 subsets, and how to develop reliable biomarkers for following the response to such therapies.

At the start of the meeting, it became clear that TNF-α would be one of the biological stars of meeting and the rest of the presentations further underlined the dominant role played by this cytokine (together with other Th1 cytokines) in the pathogenesis of autoimmune diseases.

Vitiligo: an idiopathic autoimmune disease

Instead of presenting any animal models, Das presented studies of his group on vitiligo and showed that this enigmatic depigmenting disease can be classified as an autoimmune disease. Das provided persuasive data suggesting that Th1 subsets and TNF-α could play a pivotal role in the destruction of melanocytes. Although vitiligo has been associated with the classically known autoimmune diseases, patients do not seem to present with classical autoimmune parameters such as antinuclear antibodies. However, it is believed that the perturbation of homeostatic immune physiology of the host, (which is maintained via the interacting APCs–T cells and B cells) precipitates this disease such as any other autoimmune disease.

One bonus point for studying vitiligo as a model autoimmune disease is that the therapeutics for melanoma, a deadly skin cancer, could possibly be designed by studying vitiligo. Against this scenario, this presentation argued that research into vitiligo should take place under an umbrella covering both autoimmunity and cancer [3].

Inflamatory mediators & lupus autoimmunity

This presentation by Rizgar A Mageed of the London School of Medicine, Queen Mary University of London (UK), described an interesting scenario with regard to the role of TNF-α in the pathogenesis of lupus diseases. In contrast to the positive effect of anti-TNF-α drugs in arthritis, blocking of TNF-α aggravates lupus disease (and skin conditions in general [4]). Lupus is a multifaceted autoimmune disease that shows defects in almost all compartments of the immune system, including the complement system of the innate chamber and prominent adaptive components. In order to unravel its complexity, studies were performed using three types of mouse lupus models; spontaneous, congenic and engineered (transgenic and knockout) models. It became clear during these studies that the proinflammatory TNF-α has an unexpected and somewhat paradoxical association with lupus. Its involvement is summarized in Box 1.

Mageed went on to show that TNF-α can exert it pathophysiological effect via various mechanisms. These include:

▪	Selective inhibition of NF-κB activation via interference into the NEMO binding domain peptide [5];
▪	T-cell receptor (TCR) signaling thus perturbing the thymocyte selection;
▪	Influencing the architecture of the spleen, thus influencing the B-cell selection. These latter studies were conducted comparatively in neonatal and adult mice.

The final conclusions are given in Box 2.

Continuing with systemic lupus erythematosus pathogenesis

Lilian Fossati–Jimack of Imperial college of London (UK), from the group of Maria Boto, dissected the pathogenetic pathway of systemic lupus erythematosus (SLE) and, in so doing, reinforced the claim that spontaneous and engineered mouse models are essential tools in elucidating the complicated pathology of autoimmune diseases.

Systemic lupus erythematosus is a multisystem autoimmune disease, characterized by the production of an extraordinary array of autoantibodies reactive with nuclear antigens. Interaction of these autoantibodies with their cognate antigens leads to widespread inflammatory injury and underlies the pathogenesis of SLE. In mice and humans, expression of autoimmunity is under complex genetic control. By analyzing the contribution of individual alleles to a multigenic trait it was possible to obtain an in vivo assessment of the impact on the immune system of severe modifications in the expression (deficiency or overproduction) of genes suspected to play a role in the development of an autoimmune response. This approach was illustrated using complement deficient animals. There is overwhelming evidence that deficiency of classical pathway complement proteins causes the development of SLE in humans and mice and, recently, it has been suggested that one of the main activities of the classical pathway is to promote the resolution of inflammation by enhancing the clearance and uptake of dying cells by macrophages. Using a series of murine models of complement deficiency and SLE the researchers found that these mice develop a lupus-like disease and have an impaired clearance of apoptotic cells. A similar phagocytic defect in macrophages derived from C1q-deficient humans, cultured in autologous serum has also been observed. This defect was rectifiable with purified human C1q. Consistent with these findings, macrophages from two lupus-prone murine strains have an impaired phagocytosis of apoptotic cells when compared with two nonautoimmune strains. However, impaired clearance of apoptotic cells is, on its own, insufficient to produce autoimmunity. The data available from knockout mice emphasize that susceptibility to an autoimmune disease might depend on many factors in addition to the defective removal of dying cells. In summary, it is clear that the traditional view of the role of complement in autoimmunity needs revision.

Connective tissue diseases

The scheduled lecture by David Abraham was replaced by Alan Holmes of Novartis.

Last, but not least, connective tissue diseases, such as scleroderma and systemic sclerosis, have complex pathogenic mechanisms encompassing host genetics, vascular manifestations, aberrant inflammation and autoimmunity, leading to enhanced tissue repair, resulting in scarring and replacement fibrosis. Contemporary approaches use reporter transgenesis to track and target pathogenic cells and knock-in and -out technologies to manipulate the cells and key molecular events involved. These are utilized within existing, naturally occurring disease models and those induced by modulating the environment. Developing useful systems to model and study human disease processes in vivo represents a major biomedical challenge, as does their interpretation and utility as preclinical models to reliably access novel therapeutics. In order to elucidate in this direction, Holmes presented an elegant lecture in which he highlighted the role of connective tissue growth factor (CTGF), an enigmatic cytokine, by emphasizing the following aspects as depicted in Box 3.

Neurological autoimmune diseases

Hermann Bohnenkamp (Miltenyi Biotec Ltd, Germany) and Stefanie Kuerten (University of Cologne, Germany) both focused on neurological diseases such as experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). These researchers also used specific sets of mouse models to understand the pathological mechanisms involved. The importance of these two presentations is that they demonstrate how diagnostic parameters, evaluated from the mouse studies, could be applied successfully to monitor patients once sufficiently sensitive and specific assays have been established. Using very elegant techniques, these authors demonstrated that it is possible to monitor changes in such multiple immune parameters in mice during disease progress, using a very small quantity of blood. The information obtained raises the hope that such diagnostic techniques will quickly be transferred to the clinic. Not surprisingly, both these talks, presented by young investigators, dominated the discussion. In this respect, both presentations merit special note.

Experimental autoimmune encephalomyelitis

The presentation by Bohnenkamp entitled, ‘Latest advances in helper T-cell subset analysis in autoimmunity‘ also emphasized the importance in autoimmune research of CD4⁺, Th1 and Th17 cells [6]. In EAE, both Th1 and Th17 cells, responsive to myelin autoantigens, appear throughout the course of the disease, but the specific roles of these cell populations, in particular the stability of their cytokine production phenotype, are still being elucidated. To pursue this question, it is vital to generate pure cell populations, producing the cytokine of interest, devoid of any contaminating cells. For this purpose, a two-color secretion assay (ELISPOT) was developed for quantifying viable IFN-γ- and IL-17-producing T cells directly from the blood and the spleen.

The principal findings also inferred that IFN-γ, produced by Th cells, plays a prominent role in RA. These effector–memory Th1 cells are abundant in inflamed tissues. However, it is not clear how these cells become activated at the site of chronic inflammation in the absence of pathogens and, thus, without stimulation of the TCR. To allow for an accurate analysis of T cells, the technique for isolating viable IFN-γ (other cytokines) secreting cells from tissue-like synovial tissues was reviewed.

Continuing on the same theme, Kuerten asked the question of whether or not changes in the neuroantigen-specific Th1/Th17 T-cell compartment is reflective of clinical disease in EAE and MS patients (for a review of the whole topic see [7]). Her results supported the concept that CD4⁺/IFN-γ and Th17 cells are the important agents in the development of EAE in the mouse. Similar models can also be applied for MS. Her thesis was ‘The magnitude of the neuroantigen-specific T-cell response is the primary variable that defines the clinical disease‘. Using the murine model, she demonstrated that there is a correlation between the neuroantigen specific IFN-γ/IL-17 response and the clinical course of EAE in a murine model, using small quantity, whole-blood samples. Until now it has not been possible to measure antigen-specific immune responses in mouse blood without sacrificing the animal. This is due to the very limited numbers of peripheral blood mononuclear cells that can be obtained from the blood of individual mice. Therefore, the question of whether central disease parameters, such as onset, progression and severity, correlate with a variable magnitude and cytokine quality of the T-cell response (IFN-γ and IL-17 concentrations) remained unanswered.

The interesting aspects of her presentation was the careful attention given to developing methodology tools sensitive and specific enough to quantify these parameters.

The investigators used the elegant ELISPOT-based peripheral blood mononuclear cell test system to assay parameters in as little as 150 µl of murine blood obtained from the tail vein. This allows mice to be bled repeatedly and longitudinally, while continuing to observe the clinical course of EAE. Having this assay at hand, the investigators followed up in evaluating the antigen-specific IFN-γ and IL-17 levels in the blood and subsequently showed that these parameters can indeed predict disease onset and further disease dynamics in the chronic disease, triggered by MOG:35–55 in the C57BL/6 strain and disease dynamic in the myelin proteolipid protein peptide 139–151-induced remitting–relapsing EAE of SJL/J mice.

The results presented raise hope that this valuable analytical tool may be used to provide more efficient diagnostic tests and identify new therapeutic options in human MS and other autoimmune diseases. In this regard, the commercial companies such as MACS (Miltenyi Biotec, Bergisch Gladbach, Germany), Cellular Technology Ltd (Bonn, Germany) and Novusci (Ridgmont, Bedfordshire, UK have antigen-specific T-cell assays, which can be exploited in future.

Concluding comments

More than 3% of the population suffer from some form of autoimmune disease and this meeting presented data using well-validated models of human autoimmunity. The emphasis of the meeting elucidated a pathogenetic role of the Th1/Th17/Treg axis on the one hand, and on the other, the importance of several cytokines such as INF-γ, IL-17, TNF-α and TGF-β, which pointed towards the development of novel therapeutic strategies. Another important aspect was that, although T-cell assays are complex and need very highly developed laboratories that can satisfy the regulatory agencies, the development of new assays means that it is now easier for companies to generate acceptable data in support of new drugs and/or ‘surrogate‘ clinical end points.

In conclusion, the availability of antigen specific T-cell assays (talks by Kuerten and Bohnenkamp) mean that, in the future, any reasonably equipped laboratory should be able to monitor the status of autoimmune patients during therapy, which will optimize patient treatment.

Box 1. Does TNF-α protect against autoantibody formation?

▪	TNF-α contributes to inflammation and tissue damage.
▪	Low TNF-α level is associated with lupus.
▪	Recombinant TNF-α ameliorates murine lupus.
▪	TNF-α blockade in rheumatoid arthritis patients induces anti-DNA autoantibodies.

Box 2. Role of TNF-α in lupus and autoimmunity.

▪	TNF-α has a central role in regulating the immune system.
▪	Blockade of TNF-α has spill-over effects resulting in excess cytokine production, precipitating the accelerated overproduction of autoantibodies inducing a lupus-type autoimmunity.
▪	Signaling through the classical pathway of NF-κB is important for the dominant regulatory effect of TNF-α.

Box 3. CTGF, a marker of, and possible target for, treating connective tissue diseases.

▪	Genetic targeting of mesenchymal cells is a robust and powerful approach to study tissue repair and scarring.
▪	Genetic manipulation of CTGF and TGF-β, which are potent activators of fibroblasts and progenitor cells, has a significant impact upon scarring and fibrosis.
▪	Overexpression of CTGF promotes spontaneous fibrosis in animal models.
▪	Conventional or global knockout animals are lethal.
▪	Targeted knockout of CTGF leads to a loss of pericytes.
▪	Targeted knockout of CTGF and antibody targeting of CTGF lead to attenuation of bleomycin-induced skin and lung fibrosis.
▪	Does CTGF facilitate TGF-β/PDGF functions or act alone?
▪	CTGF is a good biomarker but is it a realistic target?

Acknowledgements

The meeting was organized by Euroscicon (www.euroscicon.com). We would like to thank all the speakers who helped make the meeting such an interesting and stimulating one and whose presentations we have so ruthlessly pruned for this meeting review.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.