Researchers from the Leeds Institute of Rheumatic and Musculoskeletal Medicine are studying autoimmune disease progression looking into the key players in interferon production. In this article, BSI member, Dr Antony Psarras, discusses their recent findings published in Nature Communications which unravel the cellular sources responsible for interferon production in patients with systemic lupus erythematosus.
Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterised by the breakdown of immune tolerance and the presence of autoantibodies against nuclear antigens. One of the most profound immune abnormalities is the aberrant activation of the type I interferon (IFN) axis, as demonstrated by increased expression of interferon-stimulated genes (ISGs) in the peripheral blood of patients with SLE.1
Our research group in the Leeds Institute of Rheumatic and Musculoskeletal Medicine, led by Dr Ed Vital, has reported that a key determinant of progression from a stage of benign preclinical autoimmunity to established clinical autoimmune disease is the level of IFN activity.2
Although the cellular source and regulation of type I IFNs in SLE had not previously been clear, plasmacytoid dendritic cells (pDCs) – the professional type I IFN-producing cells – were thought to be dysregulated and contribute to excessive type I IFN production, in turn activating other compartments of the immune system such as T cells.
Therefore, one of the key questions when I started my PhD was to address why pDCs are functionally impaired and how they contribute to early stages of disease pathogenesis.
Characterising the role of pDCs in preclinical autoimmunity and SLE
Peripheral blood pDCs were enumerated and immunophenotyped by flow cytometry from healthy controls, patients with SLE and a unique cohort of antinuclear antibody (ANA)-positive individuals, who did not fulfil criteria for an established autoimmune disease and were defined as ‘At-Risk’. We found that circulating pDC numbers were significantly reduced in SLE independently of critical parameters such as type I IFN activity in peripheral blood (IFN Score), disease activity, treatment with immunosuppressant agents and corticosteroids. More interestingly, this finding was also extended to the At-Risk individuals who did not exhibit any clinical symptoms, were treatment naïve, and usually never developed clinical autoimmunity in follow-up.
We found that circulating pDC numbers were significantly reduced in SLE independently of critical parameters such as type I IFN activity in peripheral blood (IFN Score), disease activity, treatment with immunosuppressant agents and corticosteroids.
Next, we sought to investigate how pDCs respond to TLR stimulation. pDCs were purified from peripheral blood and were cultured in vitro in the presence of TLR9 or TLR7 agonists before they were analysed by flow cytometry for their cytokine-producing capacity. Unlike the pDCs from healthy controls, which responded to the TLR stimulation by producing both IFN-a and TNF, the pDCs from patients with SLE and At-Risk individuals failed to produce a significant cytokine response, independently of their intracellular expression of TLR9 and TLR7.
Since pDCs possess weak antigen presenting properties, we also co-cultured pDCs with allogeneic naïve CD4+ T cells to assess their capacity to induce T cell responses4. pDCs from both SLE and At- Risk individuals failed to induce strong T cell proliferation and activation, while they showed significantly reduced antigen uptake compared with healthy pDCs. Additionally, while healthy pDCs enhanced cytokine responses by T cells, pDCs from SLE and At-Risk individuals actively inhibited T cell cytokine production.
Utilising transcriptomics – a picture of immune senescence
RNA-sequencing analysis of purified pDCs from healthy individuals, patients with SLE, and At-Risk individuals showed that pDCs clustered according to ISG expression (IFN Score). Samples were thus assigned into an IFN-low or an IFN-high subgroup. pDCs from SLE and At-Risk individuals had a range of IFN scores but an overall higher IFN Score than pDCs from healthy individuals. Given that the impaired function of pDCs seen in preclinical and established autoimmunity was independent of IFN Score, we investigated the differentially expressed genes in pDCs from patients in both the IFN-high and IFN-low subgroups compared with pDCs from healthy individuals. We found 80 commonly expressed transcripts corresponding to cellular senescence and stress pathways. Further in vitro experiments confirmed that SLE pDCs had increased telomere erosion, while mild oxidative stress could strongly interfere with type I IFN responses.
Further in vitro experiments confirmed that SLE pDCs had increased telomere erosion, while mild oxidative stress could strongly interfere with type I IFN responses.
Keratinocytes take control – a key player in type I IFN production
If pDCs are functionally impaired in SLE and preclinical autoimmunity, then what is the cellular source of type I IFNs? Skin is the most commonly affected tissue in SLE. We noticed that IFN Score was particularly enriched in skin biopsies of SLE and At-Risk individuals, even in the absence of cutaneous inflammation. As a result, we used in situ hybridisation in the same skin biopsies to visualise the expression of type I IFNs. Interestingly, we found a diffuse expression of IFNK in the epidermis without leukocyte infiltration.
Further in vitro analysis of isolated keratinocytes from these biopsies confirmed that these cells were primed to produced type I IFNs, whereas they demonstrated a stronger response to TLR3 and RIG-I agonists compared with keratinocytes from healthy individuals.
Our findings have recently been published in Nature Communications.3 We can conclude that non-haematopoietic tissues such as the skin are not passive targets for leucocyte mediated immune processes but have an active role in generating an IFN response, which dominates over functionally exhausted pDCs, and that this is present prior to disease initiation.
So-called ‘target’ organs may therefore play an active role in the autoimmune process, which may explain the resistance of tissue inflammation to commonly used therapies that target leucocytes in SLE, pointing to therapeutic targets that lie outside the conventional immune system. Future work may offer further insights into the early events in the initiation of autoimmunity and perpetuation of inflammatory responses.
Antony Psarras MD MSc PhD
Internal Medicine Trainee, King’s College Hospital, NHS Foundation Trust