Determining integrin-mediated regulation of dendritic cell function in rheumatoid arthritis

Lead supervisor - Dr Vicky Morrison, University of Glasgow, Dr Catharien Hilkens, Newcastle University

Additional supervisors - Prof Iain McInnes, University of Glasgow; Dr Andrew Filer, University of Birmingham

Aims

This project will build on our previous (1) and ongoing studies in mouse model systems which have identified a central role for integrins in immune modulation. It will critically evaluate the functional contribution of integrin-mediated immune regulation of human dendritic cells (DCs) to RA pathogenesis. Three key aims will be addressed

1. To determine beta2 integrin expression levels and activation status (conformation) in DCs and other myeloid cells in the inflamed joint
2. To thereafter examine their ability to initiate downstream immuno-modulatory signaling pathways
3. To translate the foregoing to define the therapeutic potential of beta2 integrin-mediated regulation of DCs in inflammatory arthritis.

Background

In healthy individuals, the immune system is tightly regulated to prevent inappropriate autoimmune responses against host tissues. For RA to develop, one or more mechanism(s) of immune regulation are disrupted, resulting in a breach of tolerance and the initiation of an autoimmune response.  Such autoimmunity in turn promulgates inflammatory arthritis.

One recently discovered mechanism of immune regulation is mediated via the beta2 integrin family of adhesion receptors. Loss of beta2 integrins can result in inappropriate inflammation and autoimmunity in mice and humans (2)(3). Importantly, deficiency in one particular beta2 integrin family member, CD11b, results in exacerbated joint pathology in a murine model of RA (4). The precise mechanisms underlying this striking phenomenon are now of intense interest as they could allude to therapeutically useful pathways.

Our recent studies have shown that loss of beta2 integrin signaling specifically in dendritic cells (DCs) results in exaggerated DC activation, which in turn leads to enhanced immune responses in mice (1). As DCs are the key programmers that control immune activation versus tolerance, integrins are likely critically involved in immune regulation and maintenance of tolerance.  We now need to interrogate human DCs to understand how integrin-mediated regulation might be involved in breach of immune tolerance and how this could promote RA pathogenesis. Targeting DCs is considered a promising approach to RA treatment (5); by understanding integrin-mediated suppression of DC function we propose to design rational, novel DC targeting modalities.

Hypothesis

We propose that immuno-modulatory integrin signalling is dysregulated in RA, particularly in DC, thereby contributing to RA pathology. Regulation of these modulatory pathways will offer novel therapeutic targets for the treatment of RA.

Experimental outline
1. To determine beta2 integrin expression levels and activation status (conformation), in DCs and other myeloid cells in the inflamed joint
Peripheral blood (PB), synovial fluid (SF) and synovial tissue (ST) samples will be collected from RA patients and healthy controls (PB only). Samples derived from patients in the early inflammatory arthritis cohort will be of particular interest although established disease will also be investigated. Expression of the beta2 integrin, and the pairing alpha subunits CD11a, b, and c, will be determined by flow cytometry (PB, SF, ST) and IHC/multi-colour confocal microscopy (ST). Activation-specific epitopes (mAb24, KIM-127) will reveal integrin activation status (i.e. conformation).  Prior studies in our laboratories6 have defined DC subset phenotypes in RA. Therefore, in parallel we will use dense immune FACS profiling to define the phenotype(s) and activation status of the DCs in which beta2 integrin expression has been characterized.

2. To thereafter examine their ability to initiate downstream immuno-modulatory signaling pathways
Integrin function in PB and SF DCs will be quantified using in vitro adhesion and migration assays. Integrin signaling in DCs will be assessed by flow cytometry for phospho-protein detection and a phospho-kinase array. Together with data obtained addressing aim 1, we will thereby define the functional importance of beta2 integrin activation and signaling in the chronic inflammatory setting of the arthritic joint, potentially highlighting novel molecular targets for therapeutic intervention.

3. To translate the foregoing to define the therapeutic potential of beta2 integrin-mediated regulation of DCs in inflammatory arthritis.

Guided by the results in aims 1 & 2, we will determine if manipulating the beta2 integrin-mediated suppressive signaling pathway in human DCs is functionally achievable. Using PB DCs from healthy individuals we will, as a proof of principle, assess the effects of switching off integrin signaling in DCs using integrin blocking antibodies7 and inhibitors of relevant candidate signaling nodes (Rac1, Syk PI3K, Akt). Then, using DCs from PB and/or SF of RA patients and guided by results from aims 1 & 2, we will determine the impact of activating integrin signaling by providing the relevant ligand (e.g. ICAM, iC3b, fibrinogen) or inducing integrin activation with an antibody7. These results will give insight into the possibility of harnessing this regulatory pathway as a treatment strategy for RA.

Techniques

Glasgow
Integrin conformation assessment by flow cytometry
Adhesion and migration assays to determine integrin function
Integrin signaling analysis by Western blot and phospho-kinase array

Newcastle
DC isolation from RA patient ST by digestion
Functional in vitro assays to determine tolerogenicity of DCs

Birmingham
Immunohistochemistry and multi-colour confocal microscopy using ST samples from RA patients

Glasgow and Newcastle
DC isolation from PB from RA patients and healthy controls
DC isolation from SF samples from RA patients
Fluorescent-activated cell sorting for DC purification
Multi-parameter flow cytometry
Bioinformatics of nanostring (Newcastle) and phospho-array (with Glasgow Polyomics) datasets

References

1. Morrison, V.L. et al. Loss of beta2-integrin-mediated cytoskeletal linkage reprogrammes dendritic cells to a mature migratory phenotype. Nature communications 5, 5359 (2014).
2. Scharffetter-Kochanek, K. et al. Spontaneous skin ulceration and defective T cell function in CD18 null mice. J Exp Med 188, 119-131 (1998).
3. D'Agata, I.D., Paradis, K., Chad, Z., Bonny, Y. & Seidman, E. Leucocyte adhesion deficiency presenting as a chronic ileocolitis. Gut 39, 605-608 (1996).
4. Watts, G.M. et al. Manifestations of inflammatory arthritis are critically dependent on LFA-1. J Immunol 174, 3668-3675 (2005).
5. Hilkens, C.M. & Isaacs, J.D. Tolerogenic dendritic cell therapy for rheumatoid arthritis: where are we now? Clin Exp Immunol 172, 148-157 (2013).
6. Jongbloed, S.L. et al. Enumeration and phenotypical analysis of distinct dendritic cell subsets in psoriatic arthritis and rheumatoid arthritis. Arthritis research & therapy 8, R15 (2006).
7. Byron, A. et al. Anti-integrin monoclonal antibodies. J Cell Sci 122, 4009-4011 (2009).