Erik Lubberts

Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice Is Mediated via the IL-23/IL-17 Axis

Topical application of imiquimod (IMQ), a TLR7/8 ligand and potent immune activator, can induce and exacerbate psoriasis, a chronic inflammatory skin disorder. Recently, a crucial role was proposed for the IL-23/IL-17 axis in psoriasis. We hypothesized that IMQ-induced dermatitis in mice can serve as a model for the analysis of pathogenic mechanisms in psoriasis-like dermatitis and assessed its IL-23/IL-17 axis dependency. Daily application of IMQ on mouse back skin induced inflamed scaly skin lesions resembling plaque type psoriasis. These lesions showed increased epidermal proliferation, abnormal differentiation, epidermal accumulation of neutrophils in microabcesses, neoangiogenesis, and infiltrates consisting of CD4+ T cells, CD11c+ dendritic cells, and plasmacytoid dendritic cells. IMQ induced epidermal expression of IL-23, IL-17A, and IL-17F, as well as an increase in splenic Th17 cells. IMQ-induced dermatitis was partially dependent on the presence of T cells, whereas disease development was almost completely blocked in mice deficient for IL-23 or the IL-17 receptor, demonstrating a pivotal role of the IL-23/IL-17 axis. In conclusion, the sole application of the innate TLR7/8 ligand IMQ rapidly induces a dermatitis closely resembling human psoriasis, critically dependent on the IL-23/IL-17 axis. This rapid and convenient model allows further elucidation of pathogenic mechanisms and evaluation of new therapies in psoriasis.

The role of T cell interleukin-17 in conducting destructive arthritis: lessons from animal models

Interleukin-17 (IL-17) is a T cell cytokine spontaneously produced by cultures of rheumatoid arthritis (RA) synovial membranes. High levels have been detected in the synovial fluid of patients with RA. The trigger for IL-17 is not fully identified; however, IL-23 promotes the production of IL-17 and a strong correlation between IL-15 and IL-17 levels in synovial fluid has been observed. IL-17 is a potent inducer of various cytokines such as tumor necrosis factor (TNF)-α, IL-1, and receptor activator of NF-κB ligand (RANKL). Additive or even synergistic effects with IL-1 and TNF-α in inducing cytokine expression and joint damage have been shown in vitro and in vivo. This review describes the role of IL-17 in the pathogenesis of destructive arthritis with a major focus on studies in vivo in arthritis models. From these studies in vivo it can be concluded that IL-17 becomes significant when T cells are a major element of the arthritis process. Moreover, IL-17 has the capacity to induce joint destruction in an IL-1-independent manner and can bypass TNF-dependent arthritis. Anti-IL-17 cytokine therapy is of interest as an additional new anti-rheumatic strategy for RA, in particular in situations in which elevated IL-17 might attenuate the response to anti-TNF/anti-IL-1 therapy.

Effect of Arthritic Synovial Fluids on the Expression of Immunomodulatory Factors by Mesenchymal Stem Cells: An Explorative in vitro Study

Background: In diseased joints, the catabolic environment results in progressive joint damage. Mesenchymal stem cells (MSCs) can have immunomodulatory effects by secreting anti-inflammatory factors. To exert these effects, MSCs need to be triggered by pro-inflammatory cytokines. To explore the potential of MSCs as a treatment for diseased joints, we studied the effect of synovial fluid (SF) from donors with different joint diseases and donors without joint pathology on the immunomodulatory capacities of human MSCs in vitro. We hypothesized that SF of diseased joints influences the immunomodulatory effects of MSCs. Materials and Methods: MSCs were cultured in medium with SF of six osteoarthritis (OA) or six rheumatoid arthritis (RA) donors and three donors without joint pathology were used as control. Gene expressions of IL-6, HGF, TNFa, TGFb1, and indoleamine 2,3-dioxygenase (IDO) were analyzed. l-kynurenine concentration in conditioned medium (CM) by MSCs with SF was determined as a measure of IDO activity by MSCs. Furthermore, the effect of CM with SF on proliferation of activated lymphocytes was analyzed. Results: Addition of SF significantly up-regulated the mRNA expression of IL-6 and IDO in MSCs. SF(OA) induced significantly higher expression of IDO than SF(control), although no difference in IDO activity of the MSCs could be shown with a l-kynurenine assay. Medium conditioned by MSCs with SF(OA or RA) suppressed activated lymphocyte proliferation in vitro more than medium conditioned by MSCs without SF or with SF(control). Discussion: SF can influence the expression of genes involved in immunomodulation by MSCs and the effect on lymphocyte proliferation. We found indications for disease-specific differences between SFs but the variation between donors, even within one disease group was high. These data warrant further research to examine the potential application of MSC therapy in arthritic joints.

IL-4 gene therapy for collagen arthritis suppresses synovial IL-17 and osteoprotegerin ligand and prevents bone erosion

Bone destruction is the most difficult target in the treatment of rheumatoid arthritis (RA). Here, we report that local overexpression of IL-4, introduced by a recombinant human type 5 adenovirus vector (Ad5E1mIL-4) prevents joint damage and bone erosion in the knees of mice with collagen arthritis (CIA). No difference was noted in the course of CIA in the injected knee joints between Ad5E1mIL-4 and the control vector, but radiographic analysis revealed impressive reduction of joint erosion and more compact bone structure in the Ad5E1mIL-4 group. Although severe inflammation persisted in treated mice, Ad5E1mIL-4 prevented bone erosion and diminished tartrate-resistant acid phosphatase (TRAP) activity, indicating that local IL-4 inhibits the formation of osteoclast-like cells. Messenger RNA levels of IL-17, IL-12, and cathepsin K in the synovial tissue were suppressed, as were IL-6 and IL-12 protein production. Osteoprotegerin ligand (OPGL) expression was markedly suppressed by local IL-4, but no loss of OPG expression was noted with Ad5E1mIL-4 treatment. Finally, in in vitro studies, bone samples of patients with arthritis revealed consistent suppression by IL-4 of type I collagen breakdown. IL-4 also enhanced synthesis of type I procollagen, suggesting that it promoted tissue repair. These findings may have significant implications for the prevention of bone erosion in arthritis.

IL-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of NF-kappa B ligand/osteoprotegerin balance.

IL-17 is a T cell-derived proinflammatory cytokine in experimental arthritis and is a stimulator of osteoclastogenesis in vitro. In this study, we report the effects of IL-17 overexpression (AdIL-17) in the knee joint of type II collagen-immunized mice on bone erosion and synovial receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB/osteoprotegerin (OPG) expression. Local IL-17 promoted osteoclastic bone destruction, which was accompanied with marked tartrate-resistant acid phosphatase activity at sites of bone erosion in cortical, subchondral, and trabecular bone. Accelerated expression of RANKL and its receptor, receptor activator of NF-κB, was found in the synovial infiltrate and at sites of focal bone erosion, using specific immunohistochemistry. Interestingly, AdIL-17 not only enhanced RANKL expression but also strongly up-regulated the RANKL/OPG ratio in the synovium. Comparison of arthritic mice from the AdIL-17 collagen-induced arthritis group with full-blown collagen-arthritic mice having similar clinical scores for joint inflammation revealed lower RANKL/OPG ratio and tartrate-resistant acid phosphatase activity in the latter group. Interestingly, systemic OPG treatment prevented joint damage induced by local AdIL-17 gene transfer in type II collagen-immunized mice. These findings suggest T cell IL-17 to be an important inducer of RANKL expression leading to loss of the RANKL/OPG balance, stimulating osteoclastogenesis and bone erosion in arthritis.

Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis.

Two distinct IL-18 neutralizing strategies, i.e. a rabbit polyclonal anti-mouse IL-18 IgG and a recombinant human IL-18 binding protein (rhIL-18BP), were used to treat collagen-induced-arthritic DBA/1 mice after clinical onset of disease. The therapeutic efficacy of neutralizing endogenous IL-18 was assessed using different pathological parameters of disease progression. The clinical severity in mice undergoing collagen-induced arthritis was significantly reduced after treatment with both IL-18 neutralizing agents compared to placebo treated mice. Attenuation of the disease was associated with reduced cartilage erosion evident on histology. The decreased cartilage degradation was further documented by a significant reduction in the levels of circulating cartilage oligomeric matrix protein (an indicator of cartilage turnover). Both strategies efficiently slowed disease progression, but only anti-IL-18 IgG treatment significantly decreased an established synovitis. Serum levels of IL-6 were significantly reduced with both neutralizing strategies. In vitro, neutralizing IL-18 resulted in a significant inhibition of TNF-alpha, IL-6, and IFN-gamma secretion by macrophages. These results demonstrate that neutralizing endogenous IL-18 is therapeutically efficacious in the murine model of collagen-induced arthritis. IL-18 neutralizing antibody or rhIL-18BP could therefore represent new disease-modifying anti-rheumatic drugs that warrant testing in clinical trials in patients with rheumatoid arthritis.

Th17 cells, but not Th1 cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin‐17A production

OBJECTIVE Both Th1 cells and Th17 cells have been recognized in rheumatoid arthritis (RA); however, it remains unclear whether Th1 cells and/or Th17 cells are involved in driving disease chronicity and destructiveness. The aim of this study was to identify and characterize the functional role of Th17 cells in early RA. METHODS Flow cytometry analysis was performed on peripheral blood mononuclear cells (PBMCs) from treatment-naive patients with early RA and age-matched healthy volunteers. PBMCs from these patients, naive T cells, and primary CCR6- Th1 cells and CCR6+ Th17 cells were sorted and cultured in the absence or presence of synovial fibroblasts from patients with early RA (RASFs), and cytokine expression and gene transcription were analyzed. In addition, tumor necrosis factor α (TNFα)- and interleukin-17A (IL-17A)-blocking experiments were performed. RESULTS In the PBMCs of treatment-naive patients with early RA, an increased fraction of IL-17A-and TNFα-producing CCR6+ Th17 cells was observed. When cocultured with RASFs, these primary Th17 cells were potent inducers of IL-6 and IL-8 and the tissue-destructive enzymes matrix metalloproteinase 1 (MMP-1) and MMP-3, whereas primary Th1 cells or naive T cells were not. Importantly, specific up-regulation of IL-17A but not TNFα or interferon-γ was observed in RASF/Th17 cell cocultures. In addition to TNFα blocking, IL-17A neutralization was required to further down-regulate Th17 activity in RASF/Th17 cell cocultures. CONCLUSION Th17 cells, but not Th1 cells, cooperated with RASFs in a proinflammatory feedback loop, revealing a potential mechanism by which human Th17 cells drive chronic destructive disease in patients with RA. Furthermore, the neutralization of IL-17A activity is essential in current anti-TNF therapies to suppress Th17 cell activity in patients with early RA and potentially other Th17 cell-mediated disorders.

1,25-Dihydroxyvitamin D3 modulates Th17 polarization and interleukin-22 expression by memory T cells from patients with early rheumatoid arthritis

OBJECTIVE To examine the immunologic mechanism by which 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) may prevent corticosteroid-induced osteoporosis in patients with early rheumatoid arthritis (RA), with a focus on T cell biology. METHODS Peripheral blood mononuclear cells (PBMCs) and CD4+CD45RO+ (memory) and CD4+CD45RO- (non-memory) T cells separated by fluorescence-activated cell sorting (FACS) from treatment-naive patients with early RA were stimulated with anti-CD3/anti-CD28 in the absence or presence of various concentrations of 1,25(OH)(2)D(3), dexamethasone (DEX), and 1,25(OH)(2)D(3) and DEX combined. Levels of T cell cytokines were determined by enzyme-linked immunosorbent assay and flow cytometry. RESULTS The presence of 1,25(OH)(2)D(3) reduced interleukin-17A (IL-17A) and interferon-gamma levels and increased IL-4 levels in stimulated PBMCs from treatment-naive patients with early RA. In addition, 1,25(OH)(2)D(3) had favorable effects on tumor necrosis factor alpha (TNFalpha):IL-4 and IL-17A:IL-4 ratios and prevented the unfavorable effects of DEX on these ratios. Enhanced percentages of IL-17A- and IL-22-expressing CD4+ T cells and IL-17A-expressing memory T cells were observed in PBMCs from treatment-naive patients with early RA as compared with healthy controls. Of note, we found no difference in the percentage of CD45RO+ and CD45RO- cells between these 2 groups. Interestingly, 1,25(OH)(2)D(3), in contrast to DEX, directly modulated human Th17 polarization, accompanied by suppression of IL-17A, IL-17F, TNFalpha, and IL-22 production by memory T cells sorted by FACS from patients with early RA. CONCLUSION These data indicate that 1,25(OH)(2)D(3) may contribute its bone-sparing effects in RA patients taking corticosteroids by the modulation of Th17 polarization, inhibition of Th17 cytokines, and stimulation of IL-4.

Toll-like receptor 2 pathway drives streptococcal cell wall-induced joint inflammation: critical role of myeloid differentiation factor 88.

The IL-1R/Toll-like receptor (TLR) superfamily of receptors has a key role in innate immunity and inflammation. In this study, we report that streptococcal cell wall (SCW)-induced joint inflammation is predominantly dependent on TLR-2 signaling, since TLR-2-deficient mice were unable to develop either joint swelling or inhibition of cartilage matrix synthesis. Myeloid differentiation factor 88 (MyD88) is a Toll/IL-1R domain containing adaptor molecule known to have a central role in both IL-1R/IL-18R and TLR signaling. Mice deficient for MyD88 did not develop SCW-induced arthritis; both joint swelling and disturbance of cartilage chondrocyte anabolic function was completely abolished. Local levels of proinflammatory cytokines and chemokines in synovial tissue washouts were strongly reduced in MyD88-deficient mice. Histology confirmed the pivotal role of MyD88 in acute joint inflammation. TLR-2-deficient mice still allow influx of inflammatory cells into the joint cavity, although the number of cells was markedly reduced. No influx of inflammatory cells was seen in joints of MyD88-deficient mice. In addition, cartilage matrix proteoglycan loss was completely absent in MyD88 knockout mice. These findings clearly demonstrated that MyD88 is a key component in SCW-induced joint inflammation. Since agonists of the Toll-like pathway are abundantly involved in both septic and rheumatoid arthritis, targeting of MyD88 may be a novel therapy in inflammatory joint diseases.

IL-17 produced by Paneth cells drives TNF-induced shock

Tumor necrosis factor (TNF) has very potent antitumor activity, but it also provokes a systemic inflammatory response syndrome that leads to shock, organ failure, and death. Here, we demonstrate that interleukin (IL)-17, a proinflammatory cytokine known to be produced mainly by activated T cells, has a critical role in this process. Antiserum against IL-17 or deletion of Il17r protected mice against a lethal TNF challenge. Serum levels of TNF-induced IL-6 and nitric oxide metabolites were significantly reduced in mice deficient in the IL-17R. TNF-induced leukocyte influx in the small intestine was reduced, and there was no injury to the small intestine. Surprisingly, electron microscopy showed that IL-17 was constitutively present in Paneth cells of the crypts. Upon TNF challenge, the intracellular pool of IL-17 in these cells was drastically reduced, suggesting rapid release of IL-17 from the granules of Paneth cells. Our findings assign a novel role for IL-17 in an acute inflammation and identify Paneth cells as a source of the IL-17 that plays a role in this process. These data indicate that innate immune cytokine responses in the local mucosa may participate in rapidly amplifying responses to systemic inflammatory challenges.