The GM-CSF/CCL17 pathway in obesity-associated osteoarthritic pain and disease in mice.

OBJECTIVES: We have previously identified a granulocyte macrophage-colony stimulating factor (GM-CSF)/C-C motif ligand 17 (CCL17) pathway in monocytes/macrophages, in which GM-CSF regulates the formation of CCL17, and it is important for an experimental osteoarthritis (OA) model. We explore here additional OA models, including in the presence of obesity, such as a requirement for this pathway. DESIGN: The roles of GM-CSF, CCL17, CCR4, and CCL22 in various experimental OA models, including those incorporating obesity (eight-week high-fat diet), were investigated using gene-deficient male mice. Pain-like behavior and arthritis were assessed by relative static weight distribution and histology, respectively. Cell populations (flow cytometry) and cytokine messenger RNA (mRNA) expression (qPCR) in knee infrapatellar fat pad were analyzed. Human OA sera were collected for circulating CCL17 levels (ELISA) and OA knee synovial tissue for gene expression (qPCR). RESULTS: We present evidence that: i) GM-CSF, CCL17, and CCR4, but not CCL22, are required for the development of pain-like behavior and optimal disease in three experimental OA models, as well as for exacerbated OA development due to obesity, ii) obesity alone leads to spontaneous knee joint damage in a GM-CSF- and CCL17-dependent manner, and iii) in knee OA patients, early indications are that BMI correlates with a lower Oxford Knee Score (r = -0.458 and p = 0.0096), with elevated circulating CCL17 levels (r = 0.2108 and p = 0.0153) and with elevated GM-CSF and CCL17 gene expression in OA synovial tissue. CONCLUSIONS: The above findings indicate that GM-CSF, CCL17, and CCR4 are involved in obesity-associated OA development, broadening their potential as targets for possible treatments for OA.

Pharmacological Inhibition of Factor XIIa Attenuates Abdominal Aortic Aneurysm, Reduces Atherosclerosis, and Stabilizes Atherosclerotic Plaques.

BACKGROUND: 3F7 is a monoclonal antibody targeting the enzymatic pocket of activated factor XII (FXIIa), thereby inhibiting its catalytic activity. Given the emerging role of FXIIa in promoting thromboinflammation, along with its apparent redundancy for hemostasis, the selective inhibition of FXIIa represents a novel and highly attractive approach targeting pathogenic processes that cause thromboinflammation-driven cardiovascular diseases. METHODS: The effects of FXIIa inhibition were investigated using three distinct mouse models of cardiovascular disease-angiotensin II-induced abdominal aortic aneurysm (AAA), an ApoE-/- model of atherosclerosis, and a tandem stenosis model of atherosclerotic plaque instability. 3F7 or its isotype control, BM4, was administered to mice (10 mg/kg) on alternate days for 4 to 8 weeks, depending on the experimental model. Mice were examined for the development and size of AAAs, or the burden and instability of atherosclerosis and associated markers of inflammation. RESULTS: Inhibition of FXIIa resulted in a reduced incidence of larger AAAs, with less acute aortic ruptures and an associated fibro-protective phenotype. FXIIa inhibition also decreased stable atherosclerotic plaque burden and achieved plaque stabilization associated with increased deposition of fibrous structures, a >2-fold thicker fibrous cap, increased cap-to-core ratio, and reduction in localized and systemic inflammatory markers. CONCLUSION: Inhibition of FXIIa attenuates disease severity across three mouse models of thromboinflammation-driven cardiovascular diseases. Specifically, the FXIIa-inhibiting monoclonal antibody 3F7 reduces AAA severity, inhibits the development of atherosclerosis, and stabilizes vulnerable plaques. Ultimately, clinical trials in patients with cardiovascular diseases such as AAA and atherosclerosis are warranted to demonstrate the therapeutic potential of FXIIa inhibition.

Pharmacokinetic/pharmacodynamic modeling for dose selection for the first-in-human trial of the activated Factor XII inhibitor garadacimab (CSL312).

Factor XII (FXII) is a serine protease involved in multiple cascades, including the kallikrein-kinin system. It may play a role in diseases in which the downstream cascades are dysregulated, such as hereditary angioedema. Garadacimab (CSL312) is a first-in-class, fully human, monoclonal antibody targeting activated FXII (FXIIa). We describe how translational pharmacokinetic (PK) and pharmacodynamic (PD) modeling enabled dose selection for the phase I, first-in-human trial of garadacimab. The PK/PD data used for modeling were derived from preclinical PK/PD and safety studies. Garadacimab plasma concentrations rose with increasing dose, and clear dose-related PD effects were observed (e.g., a mechanism-based prolongation of activated partial thromboplastin time). The PK/PD profile from cynomolgus monkeys was used to generate minimal physiologically-based pharmacokinetic (mPBPK) models with target-mediated drug disposition (TMDD) for data prediction in cynomolgus monkeys. These models were later adapted for prediction of human data to establish dose selection. Based on the final mPBPK model with TMDD and assuming a weight of 70 kg for an adult human, a minimal inhibition (<10%) of FXIIa with a starting dose of 0.1 mg/kg garadacimab and a near maximal inhibition (>95%) at 10 mg/kg garadacimab were predicted. The phase I study is complete, and data on exposure profiles and inhibition of FXIIa-mediated kallikrein activity observed in the trial support and validate these simulations. This emphasizes the utility and relevance of translational modeling and simulation in drug development.

A cytotoxic anti-IL-3Rα antibody targets key cells and cytokines implicated in systemic lupus erythematosus.

To date, the major target of biologic therapeutics in systemic lupus erythematosus (SLE) has been the B cell, which produces pathogenic autoantibodies. Recently, targeting type I IFN, which is elaborated by plasmacytoid dendritic cells (pDCs) in response to endosomal TLR7 and TLR9 stimulation by SLE immune complexes, has shown promising results. pDCs express high levels of the IL-3Rα chain (CD123), suggesting an alternative potential targeting strategy. We have developed an anti-CD123 monoclonal antibody, CSL362, and show here that it affects key cell types and cytokines that contribute to SLE. CSL362 potently depletes pDCs via antibody-dependent cell-mediated cytotoxicity, markedly reducing TLR7, TLR9, and SLE serum-induced IFN-α production and IFN-α-upregulated gene expression. The antibody also inhibits TLR7- and TLR9-induced plasmablast expansion by reducing IFN-α and IL-6 production. These effects are more pronounced than with IFN-α blockade alone, possibly because pDC depletion reduces production of other IFN subtypes, such as type III, as well as non-IFN proinflammatory cytokines, such as IL-6. In addition, CSL362 depletes basophils and inhibits IL-3 signaling. These effects were confirmed in cells derived from a heterogeneous population of SLE donors, various IFN-dependent autoimmune diseases, and healthy controls. We also demonstrate in vivo activity of CSL362 following its s.c. administration to cynomolgus monkeys. This spectrum of effects provides a preclinical rationale for the therapeutic evaluation of CSL362 in SLE.

Monoclonal antibody targeting of IL-3 receptor α with CSL362 effectively depletes CML progenitor and stem cells.

Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) in eliminating differentiated chronic myeloid leukemia (CML) cells, recent evidence suggests that leukemic stem and progenitor cells (LSPCs) persist long term, which may be partly attributable to cytokine-mediated resistance. We evaluated the expression of the interleukin 3 (IL-3) receptor α subunit (CD123), an established marker of acute myeloid leukemia stem cells, on CML LSPCs and the potential of targeting those cells with the humanized anti-CD123 monoclonal antibody CSL362. Compared with normal donors, CD123 expression was higher in CD34(+)/CD38(-) cells of both chronic phase and blast crisis CML patients, with levels increasing upon disease progression. CSL362 effectively targeted CML LSPCs by selective antibody-dependent cell-mediated cytotoxicity (ADCC)-facilitated lysis of CD123(+) cells and reduced leukemic engraftment in mice. Importantly, not only were healthy donor allogeneic natural killer (NK) cells able to mount an effective CSL362-mediated ADCC response, but so were CML patients' autologous NK cells. In addition, CSL362 also neutralized IL-3-mediated rescue of TKI-induced cell death. Notably, combination of TKI- and CSL362-induced ADCC caused even greater reduction of CML progenitors and further augmented their preferential elimination over normal hematopoietic stem and progenitor cells. Thus, our data support the further evaluation of CSL362 therapy in CML.

House dust mite sublingual immunotherapy: the role for transforming growth factor-beta and functional regulatory T cells.

RATIONALE: Sublingual allergen-specific immunotherapy is gaining popularity for treatment of allergic diseases, but underlying immunological mechanisms are unresolved. OBJECTIVES: To perform detailed immunological investigation of sublingual house dust mite (HDM) immunotherapy. METHODS: A 12-month randomized double-blind placebo-controlled study of sublingual HDM immunotherapy in 30 HDM-allergic subjects was performed, with 1-year open extension in 9 patients on active treatment. HDM-stimulated blood mononuclear cells were analyzed for proliferation, cytokines, and regulatory T cells (Tregs) by flow cytometry and ELISA. Effects of blocking transforming growth factor (TGF)-beta and IL-10 on proliferation were determined. Treg suppressor function and allergen-specific antibody levels were measured. Clinical efficacy was assessed by symptom, medication, and Juniper quality-of-life scores. MEASUREMENTS AND MAIN RESULTS: Allergen-induced CD4(+) T-cell division and IL-5 production were significantly decreased after 6- and 12-months' active treatment but not placebo. sTGF-betaRII blocked immunotherapy-induced suppression of allergen-specific T-cell proliferation, maximal at 6 months. Decreased allergen-specific CD4(+) T-cell proliferation and increased IL-10 secretion and serum Der p 2-specific IgG(4) were maximal at 24 months' active treatment. Treg (CD4(+)CD25(+)CD127(lo)/Foxp3(+)) function was demonstrated by suppression of allergen-specific effector T-cell (CD4(+)CD25(-)CD127(hi)) proliferation and cytokine production. Clinical efficacy of immunotherapy was supported by significantly decreased rhinitis symptom score, total asthma score, and Juniper quality-of-life score. CONCLUSIONS: This study establishes the novel finding that TGF-beta mediates the immunological suppression seen early in clinically effective sublingual HDM immunotherapy in addition to an increase in Tregs with suppressor function. Clinical trial registered with www.clinicaltrials.gov (NCT00250263).

Reversing the autoimmune condition: experience with experimental autoimmune gastritis.

Autoimmune diseases remain a significant health problem in our society, despite the best efforts to understand and treat these conditions. Current clinical treatments are aimed at alleviating the consequences of these diseases, with limited prospects for cure. Our studies with the experimental model of autoimmune gastritis have led us to explore potential curative strategies that can reverse the autoimmune condition. Using mouse models, we have shown that expression of the known gastric autoantigen in the thymus results in immunological tolerance and resistance to the induction of autoimmune gastritis. Also, induced tolerance in donor mice can be transferred to syngeneic recipient mice by bone marrow cells. Strategies based on these observations could lead to reversal of established disease. Transfer of ensuing knowledge to the cure of serious human autoimmune diseases is our ultimate goal.

Tolerance established in autoimmune disease by mating or bone marrow transplantation that target autoantigen to thymus.

Autoimmune diseases are a significant cause of death and morbidity, affecting up to 5% of the population. At present, there is no cure. Autologous bone marrow transplantation has been promoted as a treatment for achieving disease reversal and long-term remission. However, clinical trials in progress in Europe and North America report a significant risk of relapse. Here, we have addressed whether we can establish tolerance in an active autoimmune disease model by thymic expression of autoantigen. We show that tolerance and disease resistance can indeed be established in transgenic mice that spontaneously develop granulocyte macrophage colony stimulating factor-induced autoimmune gastritis, by mating them with disease-resistant transgenic mice that target autoantigen to the thymus. T cells from these double-transgenic mice are non-responsive to gastric antigen in vitro and fail to initiate disease following transfer to naive recipients. Further, we show that transplantation with bone marrow from disease-resistant transgenic mice renders recipient mice with gastritis tolerant to autoantigen as shown by a dramatic fall in autoantibody levels and T cell non-responsiveness to antigen in vitro. We suggest that genetically modified bone marrow targeting autoantigen to the thymus may be used to establish tolerance and prevent relapse of autoimmune disease following autologous bone marrow transplantation.

Organ-specific autoimmunity in granulocyte macrophage-colony stimulating factor (GM-CSF) deficient mice.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a pleiotrophic proinflammatory cytokine that augments adaptive immunity by linking it to innate immunity. Experimental autoimmune gastritis is an animal model of human autoimmune gastritis and pernicious anaemia. We have previously shown that GM-CSF is expressed in the gastric mucosa of mice with gastritis initiated by neonatal thymectomy (Gastroenterology 110 (1996) 1791) and that transgenic expression of GM-CSF in the stomach induces autoimmune gastritis in mice (J. Immunol. 166 (2001) 2090). Here we have examined the development of autoimmune gastritis initiated by immunisation or by neonatal thymectomy in GM-CSF deficient mice. We found that gastritis develops in GM-CSF deficient mice initiated by neonatal thymectomy but not by immunisation with gastric antigen. These observations suggest that GM-CSF is not absolutely required for the initiation of autoimmunity and highlights the different conclusions that can be drawn using different disease models.

Animal models of human disease: experimental autoimmune gastritis--a model for autoimmune gastritis and pernicious anemia.

Human autoimmune gastritis is an organ-specific autoimmune disease of the stomach. It is characterized by the development of disease-specific autoantibodies and a pathology that specifically targets specialized cells within the gastric environment. The autoantigens associated with this disease have been defined as the gastric H+/K+ ATPase and intrinsic factor. The development of experimental disease models has been pivotal in our contemporary understanding of autoimmunity. Here we review mouse models of autoimmune gastritis and their relevance to human autoimmune gastritis associated with pernicious anemia. We appraise some historical as well as recent studies of experimental autoimmune gastritis (EAG), highlighting key findings that have formed the basis of our current understanding of the etiology and mechanism(s) associated with autoimmune gastritis. A precise understanding of the pathogenesis of autoimmune gastritis will permit the design of innovative and rational therapeutic strategies to prevent, arrest, ameliorate or reverse the disease.