Silke Frey

The novel cytokine interleukin-36α is expressed in psoriatic and rheumatoid arthritis synovium

Background Interleukin (IL)-36α is a recently described member of the IL-1 cytokine family with pro-inflammatory and clearly pathogenic properties in psoriasis. Objective To determine the IL-36α expression in psoriatic arthritis (PsA) compared to rheumatoid arthritis (RA) and osteoarthritis (OA). Methods Synovial tissues obtained from arthritis patients were stained for IL-36α, IL-36 receptor (IL-36R) and IL-36R antagonist (IL-36Ra) by immunohistochemistry and immunofluorescence. Lysates were examined for IL-36α by western blot analysis. Synovial fibroblasts (FLS) cultured in the presence of IL-36α were assayed for cytokine expression by quantitative real time PCR and multiplex assay. IL-36α-induced signal transduction in FLS was analysed by immunoblotting. Results Expression of IL-36R and its ligands IL-36α and IL-36Ra was detected in the synovial lining layer and cellular infiltrates of patients with inflammatory arthritis. IL-36α was expressed significantly higher in PsA and RA than in OA synovium. CD138-positive plasma cells were identified as the main cellular source of IL-36α. No differences were observed for the expression of IL-36R and IL-36Ra between PsA, RA and OA. Functionally, IL-36α induced the expression of IL-6 and IL-8 in FLS through p38/NFkB activation. Conclusions IL-36α is up-regulated in PsA and RA synovium, expressed by tissue plasma cells and leads to IL-6 and IL-8 production by synovial fibroblasts. Hence, IL-36α links plasma cells to inflammatory cytokine production by FLS and may represent a key link between autoimmunity and the induction of synovitis.

High frequency of autoantibody-secreting cells and long-lived plasma cells within inflamed kidneys of NZB/W F1 lupus mice

Autoantibodies to double‐stranded (ds) DNA represent a serological hallmark of systemic lupus erythematosus (SLE) and may critically contribute to the pathogenesis of lupus nephritis. Self‐reactive antibodies might be partially produced by long‐lived plasma cells (PCs), which mainly reside within the bone marrow and spleen. In contrast to short‐lived PCs, long‐lived PCs are extremely resistant to therapy and may sustain refractory disease courses. Recently, antibody‐secreting cells were found within the inflamed kidneys of New Zealand black/white (NZB/W) F1 lupus mice as well as of patients with SLE. To analyze the longevity of the IgG‐producing cells present in nephritic kidneys of NZB/W F1 mice we performed in vivo BrdU‐labeling. We identified a higher frequency of long‐lived than short‐lived renal PCs, indicating that survival niches for long‐lived PCs also exist within inflamed kidneys. Using ELISPOT assays, we found that on average 31% of renal IgG‐producing cells reacted with dsDNA and 24% with nucleolin. Moreover, the frequencies of IgG‐secreting cells specific for the autoantigens dsDNA and nucleolin were higher in the kidneys compared with those in the spleen and bone marrow.

Low dose ionising radiation leads to a NF-κB dependent decreased secretion of active IL-1β by activated macrophages with a discontinuous dose-dependency

Abstract Purpose: Therapy with low doses of ionising radiation (X-rays) exerts anti-inflammatory effects. Little is known about whether and how low doses of X-ray treatment modulate the inflammatory phenotype of macrophages, especially the secretion of Interleukin-1beta (IL-1β). Materials and methods: Macrophages were differentiated from human THP-1 monocytes, activated with lipopolysaccharide (LPS), treated with distinct low doses of X-rays, and co-activated with monosodium urate crystals (MSU) to induce inflammasome activation. Secretion of IL-1β was analysed by an enzyme-linked immunosorbent assay (ELISA) and Western blot. Furthermore, we analysed the intracellular amounts of the serine/threonine protein kinase B (named: Akt), mitogen-activated protein kinase p38 (p38), the v-rel reticuloendotheliosis viral oncogene homolog A (RelA), and pro- and cleaved IL-1β. Results: Low dose X-rays led to decreased secretion of active IL-1β in a manner discontinuous with dose which was most pronounced after 0.5 or 0.7 Gy. Passive release of lactate dehydrogenase (LDH) was not influenced by X-rays. The decreased secretion of IL-1β correlated with reduced translocation of RelA, being part of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) complex, into the nucleus. After 0.5 or 0.7 Gy of X-rays, the intracellular protein amounts of up (p38) and downstream molecules (Akt) of NF-κB were reduced in activated macrophages, as were the pro- and cleaved forms of IL-1β. Conclusions: Distinct low doses of X-rays induce an anti-inflammatory phenotype of activated macrophages by lowering the amount of secreted IL-1β in a NF-κB dependent manner.

Toll‐like Receptor 2 Is Required for Autoantibody Production and Development of Renal Disease in Pristane‐Induced Lupus

OBJECTIVE The mechanisms involved in breaking immunologic tolerance against nuclear autoantigens in systemic lupus erythematosus (SLE) are not fully understood. Our recent studies in nonautoimmune mice provided evidence of an important role of Toll-like receptor 2 (TLR-2) in antichromatin autoantibody induction by high mobility group box chromosomal protein 1-nucleosome complexes derived from apoptotic cells. The objective of this study was to investigate whether TLR-2 signaling is required for the induction of autoantibodies and the development of SLE-like disease in murine pristane-induced lupus. METHODS Lupus-like disease in C57BL/6 and TLR-2(-/-) mice was induced by pristane injection. The numbers of immune cells and serum cytokine concentrations were determined by flow cytometry. Renal disease was assessed by quantification of proteinuria, histologic analyses, and enzyme-linked immunospot assay. RESULTS Pristane-injected TLR-2(-/-) mice generated reduced numbers of splenic CD138+/cytoplasmic κL/λL chain-positive plasma cells and displayed diminished IgG responses against double-stranded DNA, histones, nucleosomes, some extractable nuclear autoantigens, and cardiolipin when compared with wild- type controls. TLR-2 deficiency prevented the pristane-induced systemic release of interleukin-6 (IL-6) and IL-10. The absence of TLR-2 attenuated peritoneal recruitment of CD11c+ cells and formation of lipogranulomas. Importantly, the renal disease that developed in pristane-treated TLR-2(-/-) mice was less severe than that in control mice, as reflected by milder proteinuria, reduced glomerular deposition of IgG and complement, and decreased renal infiltration of autoantibody-secreting cells. CONCLUSION TLR-2 is required for the production of prototypical lupus autoantibodies and the development of renal disease in pristane-induced murine lupus. Interference with TLR-2 signaling may be a promising novel strategy for the treatment of SLE.

Blockade of IL-36 Receptor Signaling Does Not Prevent from TNF-Induced Arthritis

Introduction Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines. Methods To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays. Results Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays. Conclusion Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.

Cell-Intrinsic NF-κB Activation Is Critical for the Development of Natural Regulatory T Cells in Mice

Background Naturally occurring CD4+CD25+Foxp3+ regulatory T (Treg) cells develop in the thymus and represent a mature T cell subpopulation critically involved in maintaining peripheral tolerance. The differentiation of Treg cells in the thymus requires T cell receptor (TCR)/CD28 stimulation along with cytokine-promoted Foxp3 induction. TCR-mediated nuclear factor kappa B (NF-κB) activation seems to be involved in differentiation of Treg cells because deletion of components of the NF-κB signaling pathway, as well as of NF-κB transcription factors, leads to markedly decreased Treg cell numbers in thymus and periphery. Methodology/Principal Findings To investigate if Treg cell-intrinsic NF-κB activation is required for thymic development and peripheral homeostasis of Treg cells we used transgenic (Tg) mice with thymocyte-specific expression of a stable IκBα mutant to inhibit NF-κB activation solely within the T cell lineage. Here we show that Treg cell-intrinsic NF-κB activation is important for the generation of cytokine-responsive Foxp3− thymic Treg precursors and their further differentiation into mature Treg cells. Treg cell development could neither be completely rescued by the addition of exogenous Interleukin 2 (IL-2) nor by the presence of wild-type derived cells in adoptive transfer experiments. However, peripheral NF-κB activation appears to be required for IL-2 production by conventional T cells, thereby participating in Treg cell homeostasis. Moreover, pharmacological NF-κB inhibition via the IκB kinase β (IKKβ) inhibitor AS602868 led to markedly diminished thymic and peripheral Treg cell frequencies. Conclusion/Significance Our results indicate that Treg cell-intrinsic NF-κB activation is essential for thymic Treg cell differentiation, and further suggest pharmacological NF-κB inhibition as a potential therapeutic approach for manipulating this process.

The novel interleukin-1 cytokine family members in inflammatory diseases.

Purpose of review This review provides an update on the new interleukin-1 (IL-1) cytokine family members in inflammatory diseases with focus on recent findings concerning the family members IL-36, IL-37, and IL-38 and their different expression patterns. Recent findings The IL-1 cytokines are known to be involved in many different inflammatory and autoimmune diseases. The latest IL-1 family members, IL-36, IL-37, and IL-38 have been shown to be differently regulated during course of disease. Studies of patients suffering from inflammatory diseases revealed that those cytokines are upregulated in the serum as well as in inflamed tissue. Both, epithelial cells and infiltrating peripheral mononuclear blood cells serve as source of the cytokines IL-36, IL-37, and IL-38 triggering different outcomes. These results could be confirmed in different mouse models and in-vitro and ex-vivo studies. Summary IL-36, IL-37, and IL-38 are involved in the pathogenesis of the inflammatory diseases psoriasis, rheumatoid arthritis, gout, systemic lupus erythematosus as well as Crohns disease. Thereby IL-36 acts proinflammatory triggering further inflammatory mediators. In contrast, IL-37 and IL-38 are upregulated to counteract. Understanding the imbalance of the IL-1 family is crucial for future therapeutics.

A8.6 Interleukin 33 skin overexpression does not induce inflammation

Background The cytokine interleukin 33 (IL-33), a member of the IL-1 family, is released in response to various types of endothelial or epithelial cell damage. IL-33 is constitutively expressed in epidermal keratinocytes, upregulated in inflamed skin and acts as an endogenous danger signal that mediates the recruitment of immune cells to sites of cellular damage. Our preliminary data have shown that psoriatic lesions express more nuclear IL-33 compared to perilesional biopsies from the same patients and mature IL-33 induces skin inflammation in a mouse model. Objectives To determine the role and function of IL-33 in skin inflammation, in particular to understand the mechanism how IL-33 contributes to the pathology and recruitment of inflammatory cells. Materials and Methods Transgenic mice with skin-specific expression of full length IL-33 were generated via pronuclear injection of K14-IL-33. This construct with IL-33 under keratin 14 promotor was tested for the functionality in vitro following the generation of the transgenic mice. For analysis control littermates with no transgene were used. K14-IL-33 mice were tested in various skin inflammation models including TLR7 agonistic stimulation with imiquimod 5%, ear injection model (rIL-23, 500 ng/ear for 14 days), and skin tape stripping. As readout we used caliper measurement of the ear thickness, skin histology H&E staining and immunohistochemistry as well as mRNA expression by quantitative RT-PCR. Results Pronuclear injection resulted in 16 pups with 2 transgenic mice expressing the IL-33 transgene. K14-IL-33 mice were born and developed normally with no obvious phenotype. Increased expression in the skin in K14-IL-33 compare to wildtype (WT) mice was detected using RT-PCR and immunohistology; as expected IL-33 was localised in the nuclei of the keratinocytes. No significant expression was measured in other organs. Due to the lack of a skin phenotype we performed different skin inflammation models in the K14-IL-33 mouse. Neither TLR7 stimulation via imiquimod, ear injection of IL-23, a pivotal cytokine for psoriasis, nor skin tape stripping as a model for chronic skin damage induced a clinical/histological change compared to littermates. Conclusions We demonstrate that local skin overexpression of the alarmin IL-33 does not induce spontaneous or triggered skin inflammation. In comparison to a published K14-IL-33 mouse with atopic inflammatory phenotype our data could not reproduce the described phenotype. These data suggest that different expression thresholds might lead to exacerbation of skin diseases.

Full Length Interleukin 33 Aggravates Radiation-Induced Skin Reaction

The interleukin (IL)-1 family member IL-33 has been described as intracellular alarmin with broad roles in wound healing, skin inflammation but also autoimmunity. Its dichotomy between full length (fl) IL-33 and the mature (m) form of IL-33 and its release by necrosis is still not fully understood. Here, we compare functional consequences of both forms in the skin in vivo, and therefore generated two lines of transgenic mice which selectively overexpress mmIL-33 and flmIL-33 in basal keratinocytes. Transgene mRNA was expressed at high level in skin of both lines but not in organs due to the specific K14 promoter. We could demonstrate that transgenic overexpression of mmIL-33 in murine keratinocytes leads to a spontaneous skin inflammation as opposed to flmIL-33. K14-mmIL-33 mice synthesize and secrete high amounts of mmIL-33 along with massive cutaneous manifestations, like increased epidermis and dermis thickness, infiltration of mast cells in the epidermis and dermis layers and marked hyperkeratosis. Using skin inflammation models such as IL-23 administration, imiquimod treatment, or mechanical irritation did not lead to exacerbated inflammation in the K14-flmIL-33 strain. As radiation induces a strong dermatitis due to apoptosis and necrosis, we determined the effect of fractionated radiation (12 Gy, 4 times). In comparison to wild-type mice, an increase in ear thickness in flmIL-33 transgenic mice was observed 25 days after irradiation. Macroscopic examination showed more severe skin symptoms in irradiated ears compared to controls. In summary, secreted mmIL-33 itself has a potent capacity in skin inflammation whereas fl IL-33 is limited due to its intracellular retention. During tissue damage, fl IL-33 exacerbated radiation-induced skin reaction.

Interleukin-36 receptor mediates the crosstalk between plasma cells and synovial fibroblasts

The IL‐1 family member IL‐36α has proinflammatory and pathogenic properties in psoriasis. IL‐36α binds to the IL‐36 receptor leading to nuclear factor kappa B/mitogen activated protein kinase mediated cytokine release. The IL‐36R antagonist prevents recruitment of IL‐1 receptor accessory protein and therefore IL‐36‐dependent cell activation. In inflamed human tissue, we previously could show that resident B cells and plasma cells (PC) express IL‐36α. Further, fibroblast‐like synoviocytes (FLS) produced proinflammatory cytokines upon IL‐36α‐stimulation. We hypothesize an IL‐36‐specific crosstalk between B cells/PCs and FLS permitting a proinflammatory B cell niche. Here, we firstly demonstrated that B cell lines and B cells from healthy donors express IL‐36α and stimulation increased IL‐36α in B cells and primary plasmablasts/PCs. Moreover, FLS respond specifically to IL‐36α by proliferation and production of matrix metalloproteinases via p38/HSP27 signaling. Importantly, IL‐36R‐deficiency abrogated IL‐36α−induced production of inflammatory mediators in FLS and changed the intrinsic FLS‐phenotype. Using an in vitro co‐culture system, we could show that IL‐36R‐deficient FLS had a limited capacity to support PC survival compared to wild‐type FLS. Hence, we demonstrated an IL‐36R‐dependent crosstalk between B cells/PCs and FLS. Our data support the concept of initiation and maintenance of a proinflammatory niche by B cells in the joints.