Adewonuola Alase

Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist

The interleukin-36 receptor antagonist (IL-36Ra) which regulates IL-36α, -β and -γ is linked to psoriatic inflammation, especially loss-of-function mutations in pustular psoriasis subtypes. As observed with other IL-1 superfamily proteins, the IL-36 members require N-terminal cleavage for full biological activity but the mechanisms of IL-36Ra activation remain poorly defined. Using different blood leukocyte and skin resident cell preparations, and recombinant proteins, we have identified that neutrophil elastase, but not other neutrophil derived proteases, cleaves IL-36Ra into its highly active antagonistic form. The activity of this processed form of IL-36Ra was confirmed in human primary dermal fibroblasts and keratinocytes and in skin equivalents. A significant dose dependent reduction of IL-36γ induced IL-8 and chemokine ligand 20 (CCL20) levels were detected following addition of the cleaved IL-36Ra compared to full length IL-36Ra. By activating IL-36Ra, the neutrophil derived protease can inhibit IL-36 induced chemokine production, including IL-8 and CCL20, and reduce further inflammatory cell infiltration. These findings strongly indicate neutrophil elastase to be a key enzyme in the biological function of IL-36Ra and that neutrophils can play a regulatory role in psoriatic inflammation with regard to balancing the pro-inflammatory activity of IL-36.

Interleukin-22 drives the proliferation, migration and osteogenic differentiation of mesenchymal stem cells: a novel cytokine that could contribute to new bone formation in spondyloarthropathies

Objectives. The SpAs are genetically and therapeutically linked to IL-23, which in turn regulates IL-22, a cytokine that has been implicated in the regulation of new bone formation in experimental models. We hypothesize that IL-22, a master regulator of stem cells in other niches, might also regulate human mesenchymal stem cell (MSC) osteogenesis. Methods. The effects of IL-22 on in vitro MSC proliferation, migration and osteogenic differentiation were evaluated in the presence or absence of IFN-&ggr; and TNF (to ascertain IL-22 activity in pro-inflammatory environments). Colorimetric XTT assay, trans-well migration assays, quantitative real-time PCR (qRT-PCR) for MSC lineage markers and osteogenesis assays were used. Results. Combined treatment of MSC with IL-22, IFN-&ggr; and TNF resulted in increased MSC proliferation (P = 0.008) and migration (P = 0.04), an effect that was not seen in cells treated with IL-22 alone and untreated cells. Osteogenic and adipogenic, but not chondrogenic, transcription factors were upregulated by IL-22 alone (P < 0.05). MSC osteogenesis was enhanced following IL-22 exposure (P = 0.03, measured by calcium production). The combination of IFN-&ggr; and TNF with or without IL-22 suppressed MSC osteogenesis (P = 0.03). Conclusion. This work shows that IL-22 is involved in human MSC proliferation/migration in inflammatory environments, with MSC osteogenesis occurring only in the absence of IFN-&ggr;/TNF. These effects of IL-22 on MSC function is a novel pathway for exploring pathological, post-inflammation osteogenesis in human SpA.

Therapeutic strategies in allergic contact dermatitis.

Due to its high prevalence, allergic contact dermatitis (ACD) has an important economic and occupational health impact on society. ACD presents as an inflammatory response to small molecules and involves both skin resident cells and activated skin infiltrating T cells. Activation of skin resident cells plays an essential role in the initial sensitization phase. A number of different pathways are crucially involved in this phase including the activation of pattern recognition receptors such as TLR, inflammasome activation and production of reactive oxygen species all of which contribute to release of cellular mediators such as IL-1 family members. Chemokines regulate steps in elicitation of adaptive T cell responses including the migration to and presentation of the contact allergen by skin derived antigen presenting cells in the draining lymph node as well as the recruitment of these activated, allergen reactive CD4+ and CD8+ cells back into the skin. The current therapeutic regimens are largely restricted to the avoidance of the contact allergen and the topical use of anti-inflammatory drugs such as glucocorticosteroids. Recent research, as highlighted by current patents, focus on the use of anti-oxidants, the induction of immunological tolerance, interference with cell signaling molecules and blocking of cytokines actively involved in ACD.

IFNλ Stimulates MxA Production in Human Dermal Fibroblasts via a MAPK-Dependent STAT1-Independent Mechanism

IFNλ is important for epidermal defense against viruses. It is produced by, and acts on, keratinocytes, whereas fibroblasts were previously considered to be unresponsive to this type III IFN. Herein we report findings revealing cell type-specific differences in IFNλ signaling and function in skin resident cells. In dermal fibroblasts, IFNλ induced the expression of myxovirus protein A (MxA), a potent antiviral factor, but not other IFN signature genes as it does in primary keratinocytes. In contrast to its effect on keratinocytes, IFNλ did not phosphorylate signal transducer and activator of transcription 1 in fibroblasts, but instead activated mitogen activated protein kinases (MAPK). Accordingly, inhibition of MAPK activation (p38 and p42/44) blocked the expression of MxA protein in fibroblasts but not in keratinocytes. Functionally, IFNλ inhibited proliferation in keratinocytes but not in fibroblasts. Moreover, IFNλ upregulated the expression of Tumor growth factor beta 1 (TGFβ1)-induced collagens in fibroblasts. Taken together, our findings identify primary human dermal fibroblasts as responder cells to IFNλ. Our study shows cutaneous cell type-specific IFN signaling and suggests that IFNλ, although important for epidermal antiviral competence, may also have a regulatory role in the dermal compartment balancing type I IFN-induced inhibition of tissue repair processes.

IL-36γ has proinflammatory effects on human endothelial cells

Interleukin‐36 cytokines are predominantly expressed by epithelial cells. Significant upregulation of epidermal IL‐36 is now a recognised characteristic of psoriatic skin inflammation. IL‐36 is known to induce inflammatory responses in dendritic cells, fibroblasts and epithelial cells. Although vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in skin inflammation, the effects of IL‐36 on endothelial cells are unexplored.

Prediction of autoimmune connective tissue disease in an at-risk cohort: prognostic value of a novel two-score system for interferon status

Objective To evaluate clinical, interferon and imaging predictors of progression from ‘At Risk’ to autoimmune connective tissue diseases (AI-CTDs). Methods A prospective observational study was conducted in At-Risk of AI-CTD (defined as antinuclear antibody (ANA) positive; ≤1 clinical systemic lupus erythematosus (SLE) criterion; symptom duration <12 months and treatment-naïve). Bloods and skin biopsy (non-lesional) were analysed for two interferon-stimulated gene expression scores previously described (IFN-Score-A and IFN-Score-B). Forty-nine healthy controls (HCs) and 114 SLE were used as negative and positive controls. Musculoskeletal ultrasound was performed. Progression was defined by meeting classification criteria for AI-CTDs at 12 months. Results 118 individuals with 12-month follow-up were included. Of these, 19/118 (16%) progressed to AI-CTD (SLE=14, primary Sjogren’s=5). At baseline, both IFN scores differed among At-Risk, HCs and SLE groups (p<0.001) and both were elevated in At-Risk who progressed to AI-CTD at 12 months versus non-progressors, to a greater extent for IFN-Score-B (fold difference (95% CI) 3.22 (1.74 to 5.95), p<0.001) than IFN-Score-A (2.94 (1.14 to 7.54); p=0.018). Progressors did not have significantly greater baseline clinical characteristics or ultrasound findings. Fold difference between At-Risk and HCs for IFN-Score-A was markedly greater in skin than blood. In multivariable logistic regression, only family history of autoimmune rheumatic disease, OR 8.2 (95% CI 1.58 to 42.53) and IFN-Score-B, 3.79 (1.50–9.58) increased the odds of progression. Conclusion A two-factor interferon score and family history predict progression from ANA positivity to AI-CTD. These interferon scores may allow stratification of individuals At-Risk of AI-CTD permitting early intervention for disease prevention and avoid irreversible organ damage.

Detection of IL-36γ through noninvasive tape stripping reliably discriminates psoriasis from atopic eczema

This report demonstrates that sampling and detection of IL-36γ protein by non-invasive tape stripping of skin lesion provides a highly sensitive and selective diagnostic for psoriatic inflammation.

OP0180 Type i interferon is produced by non-haematopoietic tissue resident cells but not pdcs in pre-clinical autoimmunity and sle

Background Systemic Lupus Erythematosus (SLE) is characterised by persistently high type I interferon (IFN) activity. Plasmacytoid dendritic cells (pDCs) produce large amounts of IFNs in viral infection, although these immunogenic properties are usually strictly regulated. In vitro, pDCs are responsive to nucleic acids and they have therefore been postulated to be the main source of type I IFNs in SLE. However, their function is not fully established in human SLE. Objectives To investigate the dysregulated IFN axis in patients with pre-clinical autoimmunity and SLE. Methods Patients with SLE who met 2012 ACR/SLICC criteria were recruited. We also recruited healthy controls (HC) and therapy-naïve individuals presenting with ANA and 1–2 clinical symptoms, but not meeting ACR/SLICC criteria, of whom 17% progressed to SLE (At-Risk). IFN activity was evaluated by measuring a score of IFN-responsive genes in the PBMCs using TaqMan. pDCs were immunophenotyped as well as studied in vitro for production of proinflammatory cytokines and induction of T cell responses using flow cytometry. pDCs were sorted and sequenced using high-sensitive RNA sequencing. IFN expression was visualised in skin biopsies using in situ hybridisation. Keratinocytes were isolated from fresh skin biopsies and cultured in vitro; IFN production was measured by qPCR and ELISA. Results Most of the SLE and At-Risk patients had increased IFN activity, which correlated with disease activity and clinical features. In contrast, circulating pDCs were decreased in both SLE and At-Risk patients and their numbers did not correlate with any clinical features or IFN status. In vitro stimulation revealed that pDCs from SLE and At-Risk patients could not produce IFN-α and TNF-α upon stimulation with TLR9 or TLR7 agonists. In addition, they induced significantly less T cell activation and proliferation compared to HC pDCs. RNA-seq data analysis showed an upregulation of IFN-responsive genes in most of the SLE and At-Risk pDCs but not transcripts of any IFN subtypes. Other upregulated pathways were related to immune regulation and senescence. Phenotypically, SLE pDCs were characterised by upregulation of regulatory receptors and increased telomeric erosion. In situ hybridization revealed high IFN expression in the epidermis but not in lymphocyte-infiltrating areas of lesional biopsies from SLE patients. High expression of IFN was also observed in epidermis of At-Risk individuals without any signs of cutaneous inflammation. In vitro stimulation of freshly isolated keratinocytes also showed a notable increase in IFN production. Conclusions In SLE, non-haematopoietic tissue resident cells are a dominant source of IFN and this is present prior to clinically overt disease. Meanwhile, the professional IFN-producing pDCs have lost their immunogenic properties. These findings suggest an important role for tissue resident cells in autoimmunity and may facilitate novel therapeutic interventions. Disclosure of Interest None declared