Franco Di Padova

Effects of AIN457, a Fully Human Antibody to Interleukin-17A, on Psoriasis, Rheumatoid Arthritis, and Uveitis

A human antibody to interleukin-17A is well tolerated and may be effective in the treatment of psoriasis, rheumatoid arthritis, and noninfectious uveitis. Stopping Inflammation in Its Tracks Inflammation—characterized by redness, swelling, and pain and derived from the Latin word inflammare (to set on fire)—is the body’s principal defense against infection and injury. Once the infection has been squelched by the immune system, the inflammatory response is usually switched off. Sometimes, however, immune cells activated during inflammation elude the “off switch,” resulting in tissue destruction and various diseases—including cancer, rheumatoid arthritis, and skin disorders such as psoriasis. Cytokines that activate immune cells are key drivers of inflammation. To address whether blocking one of these cytokines, interleukin-17A (IL-17A), might be a useful therapeutic strategy for treating inflammatory diseases, Hueber and colleagues used a human monoclonal antibody (AIN457) against IL-17A to treat patients in three small proof-of-concept trials for psoriasis, rheumatoid arthritis, and uveitis (eye inflammation). Their results demonstrate that IL-17A participates in these diseases and that the antibody against this cytokine may be an effective therapeutic agent. The proinflammatory cytokine IL-17A is produced by T helper 17 (TH17) cells and affects many different cell types including macrophages and dendritic cells of the immune system, as well as epithelial, endothelial, and skin cells. IL-17A has been implicated in psoriasis, rheumatoid arthritis, and uveitis, but its exact role is unclear. The etiologies and symptoms of these three diseases are very different. TH17 and TH1 cells have been implicated in both psoriasis (characterized by excessive turnover of skin cells resulting in scaly skin patches) and uveitis (intraocular inflammation that can lead to vision loss). In contrast, in the autoimmune disease rheumatoid arthritis, autoreactive T and B cells together with autoantibodies promote prolonged inflammation, ultimately resulting in the destruction of cartilage and bone. In their three proof-of-concept trials, Hueber and co-workers treated a total of 60 patients with the human monoclonal antibody AIN457 at different doses and observed no major adverse effects. Although the trials were small and the results were preliminary, improvements were seen in all three disease groups. Psoriasis patients receiving AIN457 showed reduced scaly skin patches, decreased production of inflammatory cytokines, and a reduction in T cells infiltrating the skin lesions compared with placebo-treated patients. After receiving infusions of AIN457, rheumatoid arthritis patients exhibited reduced inflammation of the synovial joints as shown by improvements in three different clinical scores compared with placebo-treated patients. Meanwhile, patients with uveitis treated with AIN457 showed improved visual acuity, reduced ocular inflammation, or a reduced need for steroid drugs after 8 weeks. These encouraging results warrant larger clinical trials to assess further the safety and efficacy of AIN457 for treating psoriasis, rheumatoid arthritis, and uveitis and perhaps other inflammatory diseases in which IL-17A has been implicated. Interleukin-17A (IL-17A) is elaborated by the T helper 17 (TH17) subset of TH cells and exhibits potent proinflammatory properties in animal models of autoimmunity, including collagen-induced arthritis, experimental autoimmune encephalomyelitis, and experimental autoimmune uveitis. To determine whether IL-17A mediates human inflammatory diseases, we investigated the efficacy and safety of AIN457, a human antibody to IL-17A, in patients with psoriasis, rheumatoid arthritis, and chronic noninfectious uveitis. Patients with chronic plaque-type psoriasis (n = 36), rheumatoid arthritis (n = 52), or chronic noninfectious uveitis (n = 16) were enrolled in clinical trials to evaluate the effects of neutralizing IL-17A by AIN457 at doses of 3 to 10 mg/kg, given intravenously. We evaluated efficacy by measuring the psoriasis area and severity index (PASI), the American College of Rheumatology 20% response (ACR20) for rheumatoid arthritis, or the number of responders for uveitis, as defined by either vision improvement or reduction in ocular inflammation or corticosteroid dose. AIN457 treatment induced clinically relevant responses of variable magnitude in patients suffering from each of these diverse immune-mediated diseases. Variable response rates may be due to heterogeneity in small patient populations, differential pathogenic roles of IL-17A in these diseases, and the different involvement or activation of IL-17A–producing cells. The rates of adverse events, including infections, were similar in the AIN457 and placebo groups. These results support a role for IL-17A in the pathophysiology of diverse inflammatory diseases including psoriasis, rheumatoid arthritis, and noninfectious uveitis.

Regulation of the MEF2 family of transcription factors by p38

ABSTRACT Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.

Blocking of Interleukin-17 during Reactivation of Experimental Arthritis Prevents Joint Inflammation and Bone Erosion by Decreasing RANKL and Interleukin-1

Rheumatoid arthritis is characterized by an intermittent course of disease with alternate periods of remission and relapse. T cells, and in particular the T-cell cytokine interleukin-17 (IL-17), are expected to be involved in arthritic flares. Here, we report that neutralizing endogenous IL-17 during reactivation of antigen-induced arthritis prevents joint inflammation and bone erosion. Synovial IL-17 mRNA expression was clearly up-regulated during primary arthritis and was further enhanced after antigen rechallenge. Neutralization of IL-17 significantly prevented joint swelling at day 1 of flare and significantly suppressed joint inflammation and cartilage proteoglycan depletion at day 4, as assessed by histology. Blocking IL-17 also clearly reduced bone erosions. Cathepsin K, a marker of osteoclast-like activity, and synovial RANKL mRNA expression were both suppressed. The degree of bone erosions strongly correlated with the severity of joint inflammation, suggesting that anti-IL-17 treatment reduced bone erosion by suppressing joint inflammation. Interestingly, blocking IL-17 suppressed synovial expression of both IL-1beta and tumor necrosis factor-alpha, whereas blocking IL-1 did not affect tumor necrosis factor-alpha levels. These data indicate that IL-17 is an important upstream mediator in joint pathology during flare-up of experimental arthritis.

The anti-CD20 antibody rituximab reduces the Th17 cell response

OBJECTIVE Rituximab has been shown to be successful in the treatment of rheumatoid arthritis (RA), and this unexpected finding indicates that B cells have an important role in this disease. The present study was undertaken to investigate the mechanism of action of rituximab in RA. METHODS Twelve patients with active RA were treated with rituximab. Disease activity was evaluated using the 28-joint Disease Activity Score. Synovial biopsy samples obtained at baseline and 12 weeks after treatment initiation were analyzed by microarray, quantitative polymerase chain reaction, and immunohistochemistry. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and from 4 patients with X-linked agammaglobulinemia were stimulated with the Th17-inducing stimulus Candida albicans, and the response in the presence and absence of rituximab was examined. RESULTS In RA patients, rituximab reduced expression of retinoic acid-related orphan receptor γt and interleukin-22 (IL-22) and numbers of Th17-positive cells in synovial tissue, and this correlated with better clinical outcome. Rituximab did not affect tumor necrosis factor α (TNFα), Th1 cell, or Treg cell responses. Rituximab strongly reduced in vitro IL-17 and IL-22 production induced by C albicans. This effect was not observed in PBMCs from patients with X-linked agammaglobulinemia. CONCLUSION Rituximab reduced the local Th17 response in RA patients, whereas it did not influence Th1 cell, Treg cell, or TNFα responses. The decreased Th17 response was associated with reduced inflammation and better clinical outcome. Moreover, inhibition of the Th17 response by rituximab was lost in the absence of B cells, providing evidence that the effects of rituximab are due to B cell depletion. These data demonstrate an unexpected role of B cells in the development of Th17 responses, which could possibly lead to B cell-based strategies for the treatment of Th17-related autoimmune diseases.

Regulation of TNF Expression by Multiple Mitogen-Activated Protein Kinase Pathways

Stimulating macrophages with bacterial endotoxin (LPS) activates numerous intracellular signaling pathways that lead to the production of TNF. In this study, we show that four mitogen-activated protein (MAP) kinase pathways are activated in LPS-stimulated macrophages: the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase, p38, and Big MAP kinase (BMK)/ERK5 pathways. Although specific activation of a single MAP kinase pathway produces only a modest effect on TNF promoter activation, activation of each MAP kinase pathway is important for full induction of the TNF gene. Interestingly, a dramatic induction of TNF promoter-driven gene expression was observed when all of the four MAP kinase pathways were activated simultaneously, suggesting a cooperative effect among these kinases. Unexpectedly, cis elements known to be targeted by MAP kinases do not play a major role in multiple MAP kinase-induced TNF gene expression. Rather, a 40-bp sequence harboring the TATA box, is responsible for the gene up-regulation induced by MAP kinases. The proximity of the MAP kinase-responsive element to the transcriptional initiation site suggested that MAP kinases regulate the transcriptional initiation complex. Utilizing α-amanitin-resistant RNA polymerase II mutants with or without a C-terminal domain (CTD) deletion, we found that deleting the CTD to 31 tandem repeats (Δ31) led to >90% reduction in MAP kinase-mediated TNF production. Thus, our data demonstrate coordination of multiple MAP kinase pathways in TNF production and suggest that the CTD of RNA polymerase II is required to execute MAP kinase signaling in TNF expression.

IL-1 and IL-23 Mediate Early IL-17A Production in Pulmonary Inflammation Leading to Late Fibrosis

Background Idiopathic pulmonary fibrosis is a devastating as yet untreatable disease. We demonstrated recently the predominant role of the NLRP3 inflammasome activation and IL-1β expression in the establishment of pulmonary inflammation and fibrosis in mice. Methods The contribution of IL-23 or IL-17 in pulmonary inflammation and fibrosis was assessed using the bleomycin model in deficient mice. Results We show that bleomycin or IL-1β-induced lung injury leads to increased expression of early IL-23p19, and IL-17A or IL-17F expression. Early IL-23p19 and IL-17A, but not IL-17F, and IL-17RA signaling are required for inflammatory response to BLM as shown with gene deficient mice or mice treated with neutralizing antibodies. Using FACS analysis, we show a very early IL-17A and IL-17F expression by RORγt+ γδ T cells and to a lesser extent by CD4αβ+ T cells, but not by iNKT cells, 24 hrs after BLM administration. Moreover, IL-23p19 and IL-17A expressions or IL-17RA signaling are necessary to pulmonary TGF-β1 production, collagen deposition and evolution to fibrosis. Conclusions Our findings demonstrate the existence of an early IL-1β-IL-23-IL-17A axis leading to pulmonary inflammation and fibrosis and identify innate IL-23 and IL-17A as interesting drug targets for IL-1β driven lung pathology.

Critical Role of Lipopolysaccharide-Binding Protein and CD14 in Immune Responses against Gram-Negative Bacteria

LPS-binding protein (LBP) and CD14 potentiate cell activation by LPS, contributing to lethal endotoxemia. We analyzed the contribution of LBP/CD14 in models of bacterial infection. Mice pretreated with mAbs neutralizing CD14 or LBP showed a delay in TNF-α production and died of overwhelming infection within 24 h, after a challenge with 250 CFU of virulent Klebsiella pneumoniae. Blockade of TNF-α also increased lethality, whereas pretreatment with TNF-α protected mice, even in the presence of LBP and CD14 blockade. Anti-LBP or anti-CD14 mAbs did not improve or decrease lethality with a higher inoculum (105 K. pneumoniae) and did not affect outcome following injections of low or high inocula of Escherichia coli O111. These results point to the essential role of LBP/CD14 in innate immunity against virulent bacteria.

Interleukin-1 drives pathogenic Th17 cells during spontaneous arthritis in interleukin-1 receptor antagonist–deficient mice

OBJECTIVE Interleukin-1 receptor antagonist-deficient (IL-1Ra-/-) mice spontaneously develop an inflammatory and destructive arthritis due to unopposed excess IL-1 signaling. In this study, the role of Th17 cells and the effect of neutralization of IL-17, IL-1, and tumor necrosis factor alpha (TNFalpha) were investigated in this IL-1-driven murine arthritis model. METHODS T cells isolated from IL-1Ra-/- and wild-type (WT) mice were stained for IL-17 and interferon-gamma, with results assessed by fluorescence-activated cell sorting analysis. To investigate the contribution of IL-1 and IL-17 in further progression of arthritis in this model, mice were treated with neutralizing antibodies after the onset of arthritis. RESULTS Compared with WT mice, IL-1Ra-/- mice had similar levels of Th1 cells but clearly enhanced levels of Th17 cells; this increase in the number of Th17 cells was evident even before the onset of arthritis, in young, nonarthritic IL-1Ra-/- mice. The percentage of Th17 cells increased even more after the onset of arthritis and, similar to the serum levels and local messenger RNA levels of IL-17, the percentage of IL-17+ Th17 cells clearly correlated with the severity of arthritis. Anti-IL-17 treatment prevented any further increase in inflammation and bone erosion, whereas blocking of TNFalpha after the onset of arthritis had no effect. In contrast, neutralization of IL-1 resulted in a complete suppression of arthritis. Interestingly, this anti-IL-1 treatment also significantly reduced the percentage of IL-17+ Th17 cells in the draining lymph nodes of these arthritic mice. CONCLUSION Increased levels of Th17 cells can be detected in IL-1Ra-/- mice even preceding the onset of arthritis. In addition, the results of cytokine-blocking studies demonstrated that IL-17 contributes to the inflammation and bone erosion in this model, which suggests that IL-1 is the driving force behind the IL-17-producing Th17 cells.

Dual Effects of p38 MAPK on TNF-Dependent Bronchoconstriction and TNF-Independent Neutrophil Recruitment in Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome

The administration of endotoxins from Gram-negative bacteria induces manifestations reminding of acute respiratory distress syndrome. p38 MAPKs have been implicated in this pathology. In this study, we show that the specific p38 α,β MAPK inhibitor, compound 37, prevents LPS-induced bronchoconstriction and neutrophil recruitment into the lungs and bronchoalveolar space in a dose-dependent manner in C57BL/6 mice. Furthermore, TNF induction and TNF signals were blocked. In TNF-deficient mice, bronchoconstriction, but not neutrophil sequestration, in the lung was abrogated after LPS administration. Therefore, TNF inhibition does not explain all of the effects of the p38 MAPK inhibitor. The p38 α,β MAPK inhibitor also prevented LPS-induced neutrophilia in TNF-deficient mice. In conclusion, LPS provokes acute bronchoconstriction that is TNF dependent and p38 MAPK mediated, whereas the neutrophil recruitment is independent of TNF but depends on LPS/TLR4-induced signals mediated by p38 MAPK.

SAR of 4-hydroxypiperidine and hydroxyalkyl substituted heterocycles as novel p38 map kinase inhibitors

The 4-hydroxypiperidine substituent was found to confer high p38 selectivity devoid of COX-1 affinity, when attached to a series of pyridinyl substituted heterocycles. Pyridinyloxazole 11 showed a promising in vivo profile with bioavailability of 64% and ED50 in rat collagen induced arthritis of 10 mg/kg po bid. In contrast to pyridinylimidazoles such as SB 203580, 11 did not inhibit human cytochrome P450 isoenzymes.