Background The objective of the study was to explore the disease pathways activated in the inflammatory foci of skin lesions in cutaneous lupus erythematosus (CLE) and dermatomyositis (DM). Methods Skin biopsies acquired from active CLE and DM lesions, patient (PC), and also healthy controls (HC) were investigated. Biopsy sections were examined by a pathologist, inflammatory foci were laser micro-dissected and captured, and proteins within captured tissue were detected in an unbiased manner by mass spectrometry. Protein pathway analysis was performed by the string-db.org platform. Findings of interest were confirmed by immunohistochemistry (IHC). Results Proteome investigation identified abundant expression of interferon-regulated proteins (IRP) as a common feature of CLE and DM. Interleukin (IL)-16 was the only abundant cytokine differentially expressed in CLE compared to DM. Caspase-3, an enzyme that cleaves IL-16 into its active form, was detected in low levels. Significantly higher proportion of IL-16- and caspase-3-positive cells was identified in CLE lesions in comparison with DM, PC, and HC. Proteomic results indicate more abundant complement deposition in CLE skin lesions. Conclusions Using unbiased mass spectrometry investigation of CLE and DM inflammatory infiltrates, we confirmed that high IRP expression is a common feature of both CLE and DM, while IL-16 is the only differentially expressed cytokine in CLE. IHC confirmed high expression of IL-16 and caspase-3 in CLE. Our novel molecular findings indicate that IL-16 detection could be useful in differential diagnostics between the two conditions that can display similar histopathological appearance. IL-16 could be of interest as a future therapeutic target for CLE.

ObjectiveLupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE). The pathogenesis is incompletely understood and diagnostic biomarkers are scarce. We investigated interleukin (IL) 16 as a potential biomarker for LN in a well-characterised cohort of patients with SLE.MethodsWe measured urinary (u-) and plasma (p-) levels of IL-16 in predefined patient groups using ELISA: LN (n=84), active non-renal SLE (n=63), inactive non-renal SLE (n=73) and matched population controls (n=48). The LN group included patients with recent biopsy-confirmed proliferative (PLN, n=47), mesangioproliferative (MES, n=11) and membranous (MLN, n=26) LN. Renal expression of IL-16 was investigated by immunohistochemistry. Associations between IL-16 measurements and clinical parameters and the diagnostic value for LN were explored.Resultsp-IL-16 was detected in all investigated cases and high p-IL-16 levels were observed in patients with active SLE. u-IL-16 was detected (dt-u-IL-16) in 47.6% of patients with LN, while only up to 17.8% had dt-u-IL-16 in other groups. In the LN group, 68% of patients with PLN had dt-u-IL-16, while the proportions in the MLN and MES groups were lower (11.5% and 45.5%, respectively). The highest u-IL-16 levels were detected in the PLN group. In the regression model, u-IL-16 levels differentiated PLN from other LN patient subgroups (area under the curve 0.775–0.896, p<0.0001). dt-u-IL-16 had superior specificity but slightly lower sensitivity than elevated anti-double-stranded DNA and low complement C3 or C4 in diagnosing PLN. A high proportion of LN kidney infiltrating cells expressed IL-16.ConclusionsWe demonstrate that detectable u-IL-16 can differentiate patients with PLN from those with less severe LN subtypes and active non-renal SLE. Our findings suggest that u-IL-16 could be used as a screening tool at suspicion of severe LN. Furthermore, the high IL-16 levels in plasma, urine and kidney tissue imply that IL-16 could be explored as a therapeutic target in SLE.

Background: The objective of the study was to explore disease pathways activated in the inflammatory foci of skin lesions in cutaneous lupus erythematosus (CLE) and dermatomyositis (DM). Methods: Skin biopsies acquired from active CLE and DM lesions, patient (PC) and also healthy controls (HC) were investigated. Biopsy sections were examined by a pathologist and inflammatory foci were laser micro-dissected, captured and proteins within captured tissue were detected in a hypothesis free manner by mass-spectrometry. Protein pathway analysis was performed by string-db.org platform. Findings of interest were confirmed by immunohistochemistry (IHC).Results: Proteome investigation identified interleukin (IL)-16 to be the only detectable and abundant cytokine differentially expressed in CLE compared to DM. Caspase-3, enzyme that cleaves IL-16 into its active form, was detected in low levels. Significantly higher proportion of IL-16 and Caspase-3 positive cells were identified in CLE lesions in comparison to DM, PC and HC. Interferon-regulated proteins (IRP) were abundant in both CLE and DM. Proteomic results indicate more abundant complement deposition in CLE skin lesions. Conclusions: Using hypothesis free mass-spectrometry investigation of CLE inflammatory infiltrates, we identified that IL-16 is the only detectable and highly abundant cytokine, while IRP was a common feature of both CLE and DM. IHC confirmed high expression of IL-16 and caspase-3 in CLE. Our novel molecular findings indicate that IL-16 detection could be useful in differential diagnostics between the two conditions that can display similar histopathological appearance. Potentially, IL-16 could be of interest as a future therapeutic target for CLE.

Background:Cutaneous lupus erythematosus (CLE) is an autoimmune disease. It can be limited to the skin or be one of manifestations of systemic LE (SLE). The typical histopathologic pattern in CLE/SLE is interface dermatitis, which can also be observed in dermatomyositis (DM). While LE may affect any organ system, DM most commonly affect muscles and skin.Objectives:The aim of this study was to investigate the whole proteome of skin inflammatory foci in the cohort of CLE and DM patients in a comparatory, hypothesis-free manner and identify disease-unique molecular mechanisms.Methods:CLE (n=6), DM (n=5) patients and controls (n=6) were recruited at diagnosis or disease exacerbation. Skin biopsies were acquired, examined by a pathologist and selected inflammatory foci were laser micro-dissected. The total protein content was analyzed by mass-spectrometry, further analysis was performed by string-db.org platform. Certain proteomic findings were confirmed by immunohistochemistry (IHC).Results:CLE infiltrates were more protein rich in comparison to DM lesions. There ratio of 5x upregulated proteins in LE/DM was 60, while ratio for DM/LE was 13. Our results confirmed high abundance of (IFN)-regulated proteins both in CLE and DM, including: IFIT, MX and OAS families. Proteins expressed differentially in CLE covered complement proteins (C1b), including membrane attack complex (MAC) (C5, C6, C7, C8A and B) and complement regulators (CFHR1, CFHR2, CFHR5), as well as regulators of coagulation: thrombospondin 2 (THBS2), thrombin (F2), fibrinogen (F12) and annexin A3 (ANXA3). Importantly, we identified interleukin (IL) -16 as the only detectable and highly abundant cytokine in the CLE lesions and confirmed this finding by IHC.Conclusion:ConclusionsOur data confirm evidence on IFN-regulated processes in CLE/SLE. Importantly, we identified IL-16 as a novel cytokine most strongly upregulated locally in the skin lesions. Moreover, we identified activation of MAC, complement regulating proteins as well as involvement of coagulation/fibrinolysis system. The study brings information on novel pathways involved in the inflammatory foci of the skin lesions in CLE patients. Our findings are of interest in further search of new therapeutic targets.Disclosure of Interests: :Timothy Niewold: None declared, Karin Popovic-Silwerfeldt: None declared, Julia Lehman: None declared, Alexander Meves: None declared, Cristine Charlesworth: None declared, Benjamin Madden: None declared, Aliisa Hayry: None declared, Aleksandra Antovic: None declared, Ingrid E. Lundberg Grant/research support from: Bristol Meyer Squibb, Corbus Pharmaceuticals, Inc and Astra Zeneca, Marie Wahren-Herlenius: None declared, Elisabet Svenungsson: None declared, Vilija Oke: None declared

BackgroundThe pro-inflammatory interleukin (IL)-16 can be released by different immune cells and has been shown to be an inflammatory mediator in rheumatic diseases. Urinary (u-) IL-16 has recently been found to be associated with proliferative nephritis in systemic lupus erythematosus (SLE) and is expressed in inflammatory foci in the kidney[1,2]. However, data on u-IL-16 in other immune-mediated kidney diseases including Antineutrophil Antibody Associated Vasculitis (AAV) is lacking.ObjectivesTo investigate levels of u- IL16 in AAV patients with kidney involvement compared to controls and to assess associations of u-IL16 with clinical variables.MethodsUrine samples from 43 AAV patients were collected at the time of kidney biopsy on clinical indication. The biopsies were classified according to histopathological class[3]. Clinical variables were extracted from medical records. Forty-eight population controls were included. The u-IL16 was measured using a commercial sandwich ELISA. The cut-off for detectable IL-16 was set to ≥ 10 pg/mL. Vasculitis disease activity at the time of kidney biopsy was assessed using the Birmingham Vasculitis Activity Score (BVAS 3.0).ResultsIn the AAV group, there were 25 males (58.1%), the median age was 61 years, and 22 (51.2%) were anti-PR3 positive while 21 (48.8%) were anti-MPO positive. There were 25 (58.1%) patients with GPA and 18 (41.9%) with MPA in the study. Median BVAS was 16 (interquartile range 13-21). Thirty-nine (90.7%) patients had biopsy-proven kidney involvement, whereas four patients had no histopathological signs of vasculitis. Mean eGFR (SD) was 51.1(31.9) in the AAV group and 91.6 (11.9) in the control group, respectively (p=<0.001). At inclusion and urine sampling 35 (81.4%) patients were on immunosuppressive treatment. In all, 15/43 (34.9%) patients had detectable levels of u-IL16, all of whom had biopsy-proven vasculitis (Figure). The AAV patients had significantly higher levels of u-IL16 compared to controls (p=0.027, 95% CI 1.79 – 27.6). Two controls had detectable levels of u-IL16. u-IL16 levels did not differ with respect to diagnosis (GPA/MPA) or ANCA-serotype. A positive correlation was seen between u-IL16 and BVAS (r=0.326, p=0.03), and there was a negative correlation between u-IL16 and eGFR (r= -0.330, p=0.03). Thirty-seven kidney biopsies were classified according to Berden: Focal (n=20), Crescentic (n=8), Mixed (n=6), and Sclerotic (n=3). There was a significant difference in u-IL16 levels between these groups (H (3)=8.68, p =0.034) with the highest proportion of patients with detectable u-IL16 in the sclerotic (66.7%) and crescentic (62.5%) classes. Levels of u-IL16 were not statistically significantly different between patients with or without ongoing treatment. The sensitivity for u-IL16 to correctly classify occurrence of kidney involvement in AAV patients was 38% (specificity 100%, PPV 100%, NPV 14%).ConclusionUrinary IL-16 was detectable in a significant proportion of AAV patients with kidney involvement and reflected disease activity and severity. As similar findings have been described in SLE, u-IL16 may be a marker of kidney inflammation in systemic immune-mediated kidney diseases. Further research is warranted to evaluate the pathogenic role of IL-16, the possible use of u-IL16 as a biomarker in clinical practice, as well as its potential as treatment target.References[1]Fava A, Rao DA, Mohan C, et al. Urine Proteomics and Renal Single-Cell Transcriptomics Implicate Interleukin-16 in Lupus Nephritis. Arthritis Rheumatol. 2022;74(5):829-839.[2]Hayry A, Faustini F, Zickert A, et al. Interleukin (IL) 16: a candidate urinary biomarker for proliferative lupus nephritis. Lupus Sci Med. 2022;9(1).[3]Berden AE, Ferrario F, Hagen EC, et al. Histopathologic classification of ANCA-associated glomerulonephritis. J Am Soc Nephrol. 2010;21(10):1628-1636.Acknowledgements:NIL.Disclosure of InterestsNone Declared.