Apoptotic signal molecules in skin biopsies of cutaneous lupus erythematosus: analysis using tissue microarray

Abstract 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.

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ID: 132: LUPUS KERATINOCYTES ARE PRIMED BY AN AUTOCRINE TYPE I INTERFERON LOOP TO ROBUSTLY SECRETE IL-6

Journal of Investigative Medicine ◽

10.1136/jim-2016-000120.136 ◽

2016 ◽

Vol 64 (4) ◽

pp. 976.2-977

Author(s):

JN Stannard ◽

TJ Reed ◽

JM Kahlenberg ◽

EM Myers ◽

L Lowe ◽

...

Keyword(s):

Lupus Erythematosus ◽

Inflammatory Cytokine ◽

Cutaneous Lupus Erythematosus ◽

Type I ◽

Healthy Controls ◽

University Of Michigan ◽

Type I Ifn ◽

Skin Biopsies ◽

The University ◽

Cutaneous Lupus

BackgroundCutaneous lupus erythematosus (CLE) is a disfiguring disease that can affect up to 70% of patients with systemic lupus. Treatment modalities are often ineffective and flares are frequent. Interleukin-6 (IL-6) is a pro-inflammatory cytokine which has gotten recent attention in SLE as IL-6 is increased in the serum of active patients and blockade of IL-6 is therapeutic in murine lupus models and phase I human trials. The source of IL-6 in CLE remains unclear.MethodsAll studies were approved by the University of Michigan Internal Review Board (IRB# 72843 and 66116 to JMK). RNA was isolated from formalin fixed, paraffin-embedded biopsies of CLE rashes, which were obtained from the University of Michigan Pathology database. Real-time PCR was used to determine the expression level of the myxovirus (influenza virus) resistance 1 (MX-1) and interleukin-6 (IL6) genes. Biopsies were stained for IL-6 using immunohistochemistry. Skin biopsies were obtained from uninvolved skin of SLE patients with a history of cutaneous involvement or healthy controls followed by isolation and culture of keratinocytes. At confluence, cultures were treated with various concentrations of TLR ligands or UVB and IL-6 release was measured via ELISA. Blockade of type I IFN signaling was completed via monoclonal antibody to the type I IFN receptor.ResultsReal-time PCR analysis of subacute cutaneous lupus erythematosus (sCLE) (n=21) and discoid (DLE) (n=22) rashes demonstrated a significant upregulation of both the IFN-regulated gene, MX1, and the pro-inflammatory cytokine IL-6 when compared with control samples (n=9). Immunohistochemical analysis of skin biopsies confirmed upregulation of IL-6 in the epidermis when compared to control. Keratinocytes from healthy skin of lupus patients produced significantly more IL-6 when stimulated by TLR2, 3 or 4 agonists or exposed to UVB radiation when compared to identical passage keratinocytes from healthy controls. Treatment of control keratinocytes with IFNα increased their IL-6 production and blockade of type I IFNs in the culture media of SKE keratinocytes downregulated the secretion of IL-6.ConclusionsIL-6 is increased at the RNA and protein level within cutaneous lupus biopsies when compared to healthy control skin. Keratinocytes are a major producer of IL-6 in the skin and lupus keratinocytes have enhanced production of IL-6 in response to TLR ligands and UV radiation. Exposure to type I IFN can increase IL-6 production in keratinocytes. SLE-derived keratinocytes downregulate IL-6 production in the presence of tonic blockade of the type I IFN receptor. These data suggest that the epidermis, which is an important barrier for environmental insults, is primed for IL-6 production by autocrine type I IFN production and that this may be one mechanism by which factors such as UV exposure may trigger rash development. Further investigations should focus on the pathogenic significance of IL-6 upregulation in the skin and whether targeting this pathway will have an impact on cutaneous disease activity.

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Thalidomide Exerts Anti-Inflammatory Effects in Cutaneous Lupus by Inhibiting the IRF4/NF-ҡB and AMPK1/mTOR Pathways

Biomedicines ◽

10.3390/biomedicines9121857 ◽

2021 ◽

Vol 9 (12) ◽

pp. 1857

Author(s):

Sandra Domingo ◽

Cristina Solé ◽

Teresa Moliné ◽

Berta Ferrer ◽

Josefina Cortés-Hernández

Keyword(s):

Side Effects ◽

Mechanism Of Action ◽

Lupus Erythematosus ◽

Immune Cell ◽

Cutaneous Lupus Erythematosus ◽

Cell Subset ◽

Th2 Response ◽

Skin Biopsies ◽

Anti Inflammatory ◽

Cutaneous Lupus

Thalidomide is effective in patients with refractory cutaneous lupus erythematosus (CLE). However, the mechanism of action is not completely understood, and its use is limited by its potential, severe side-effects. Immune cell subset analysis in thalidomide’s CLE responder patients showed a reduction of circulating and tissue cytotoxic T-cells with an increase of iNKT cells and a shift towards a Th2 response. We conducted an RNA-sequencing study using CLE skin biopsies performing a Therapeutic Performance Mapping System (TMPS) analysis in order to generate a predictive model of its mechanism of action and to identify new potential therapeutic targets. Integrating RNA-seq data, public databases, and literature, TMPS analysis generated mathematical models which predicted that thalidomide acts via two CRBN-CRL4A- (CRL4CRBN) dependent pathways: IRF4/NF-ҡB and AMPK1/mTOR. Skin biopsies showed a significant reduction of IRF4 and mTOR in post-treatment samples by immunofluorescence. In vitro experiments confirmed the effect of thalidomide downregulating IRF4 in PBMCs and mTOR in keratinocytes, which converged in an NF-ҡB reduction that led to a resolution of the inflammatory lesion. These results emphasize the anti-inflammatory role of thalidomide in CLE treatment, providing novel molecular targets for the development of new therapies that could avoid thalidomide’s side effects while maintaining its efficacy.

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THU0224 SKIN PROTEOME INVESTIGATION IN CUTANEOUS LUPUS ERYTHEMATOSUS (CLE) REVEALS NOVEL UNIQUE DISEASE PATHWAYS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.5948 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 339.2-339

Author(s):

T. Niewold ◽

K. Popovic-Silwerfeldt ◽

J. Lehman ◽

A. Meves ◽

C. Charlesworth ◽

...

Keyword(s):

Lupus Erythematosus ◽

Molecular Mechanisms ◽

Cutaneous Lupus Erythematosus ◽

Membrane Attack Complex ◽

Skin Lesions ◽

Total Protein Content ◽

Skin Biopsies ◽

Annexin A3 ◽

Protein Rich ◽

Cutaneous Lupus

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

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