Epithelial TNF Receptor Signaling Promotes Mucosal Repair in Inflammatory Bowel Disease.

TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn's disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/ß-catenin-mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/ß-catenin signaling correlates with inflammation status. TNF-deficient (Tnf-/-) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM-derived cells and TNFR expression by radioresistant IECs. Wild-type→Tnfr1/2-/- BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/ß-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/ß-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced ß-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/ß-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.

PD-L1- and calcitriol-dependent liposomal antigen-specific regulation of systemic inflammatory autoimmune disease.

Autoimmune diseases resulting from MHC class II-restricted autoantigen-specific T cell immunity include the systemic inflammatory autoimmune conditions rheumatoid arthritis and vasculitis. While currently treated with broad-acting immunosuppressive drugs, a preferable strategy is to regulate antigen-specific effector T cells (Teffs) to restore tolerance by exploiting DC antigen presentation. We targeted draining lymph node (dLN) phagocytic DCs using liposomes encapsulating 1α,25-dihydroxyvitamin D3 (calcitriol) and antigenic peptide to elucidate mechanisms of tolerance used by DCs and responding T cells under resting and immunized conditions. PD-L1 expression was upregulated in dLNs of immunized relative to naive mice. Subcutaneous administration of liposomes encapsulating OVA323-339 and calcitriol targeted dLN PD-L1hi DCs of immunized mice and reduced their MHC class II expression. OVA323-339/calcitriol liposomes suppressed expansion, differentiation, and function of Teffs and induced Foxp3+ and IL-10+ peripheral Tregs in an antigen-specific manner, which was dependent on PD-L1. Peptide/calcitriol liposomes modulated CD40 expression by human DCs and promoted Treg induction in vitro. Liposomes encapsulating calcitriol and disease-associated peptides suppressed the severity of rheumatoid arthritis and Goodpasture's vasculitis models with suppression of antigen-specific memory T cell differentiation and function. Accordingly, peptide/calcitriol liposomes leverage DC PD-L1 for antigen-specific T cell regulation and induce antigen-specific tolerance in inflammatory autoimmune diseases.

MRI and quantification of draining lymph node function in inflammatory arthritis.

While erosion and tissue necrosis are the end-stage result of inflammatory arthritis, factors that can predict their initiation and severity are unknown. In an effort to identify these prognostic factors we developed contrast-enhanced (CE)-magnetic resonance imaging (MRI) for the mouse knee to assess the pathogenesis of inflammatory arthritis. Using this approach to study synovitis and draining lymph node (LN) function we first demonstrated that the LNs of TNF-Tg mice at 5 months are significantly larger and have greater enhancement in comparison to wild-type (WT) mice. This difference correlated with the abundance of dilated LYVE-1+ sinuses in the draining LNs. Dynamic CE-MRI further demonstrated differences between TNF-Tg and WT mice in the kinetics of LN enhancement. We established an LN capacity (LNcap) measurement that is a function of both volume and CE. We demonstrated that TNF-Tg mice have a 15-fold increase over WT levels at 5 months age (P < 0.001). Amelioration of arthritis with anti-TNF therapy resulted in a significant decrease in LNcap (P < 0.0001) that approached WT levels within 4 weeks. Interestingly, this functional decrease was not associated with a reduction of lymphatic vessels, which persist after therapy in both LNs and synovium. To assess the relationship between draining LN function and synovitis, a regression analysis was performed that demonstrated a significant negative correlation (R(2) = 0.63, P = 0.01) between LNcap and synovial volume. TNF-Tg mice with a lower LNcap display an accelerated progression of arthritis. These results indicate a protective function of enhanced lymphatic drainage in inflammatory arthritis.

An anti-tumor necrosis factor-alpha antibody inhibits the development of experimental skin tumors.

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) was originally considered to have activity against malignant disease. However, recent studies suggest TNF-alpha may also act as an endogenous tumor promoter. In the present work, mice deficient in TNF-alpha either genetically (TNF-alpha(-/-)) or after blockade with a neutralizing antibody (cV1q) were used to investigate the role of TNF-alpha in skin tumor development. Papillomas were induced in wild-type (wt) mice after treatment of skin with the initiating agent 9,10-dimethyl-1,2-benzanthracene followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 15 weeks. TNF-alpha(-/-) mice were resistant to papilloma development when compared with wt mice on C57Bl/6J, 129/SvEv, and BALB/c genetic backgrounds. Primary murine keratinocytes (newborn keratinocytes) and skin homogenates were used to characterize TPA-stimulated TNF-alpha expression. TPA induced TNF-alpha protein in newborn keratinocytes in vitro and epidermis in vivo. Neutralization of TNF-alpha protein with cV1q in vivo for 0-15 weeks of promotion significantly decreased skin tumor development after 9,10-dimethyl-1,2-benzanthracene/TPA treatment. cV1q treatment during the early stages of tumor promotion (0-6 weeks) was equally effective. These data suggest that early induction of TNF-alpha is critical for skin tumor promotion. cV1q also reduced TPA-stimulated expression of matrix metalloproteinase 9 and granulocyte macrophage colony-stimulating factor, proteins that are differentially regulated in wt and TNF-alpha(-/-) epidermis. Treatment of the 410.4 transplantable breast carcinoma with cV1q reduced tumor growth in vivo, illustrating that inhibition of tumor growth through neutralization of TNF-alpha is not limited to skin carcinogenesis. These results provide further evidence for procancer actions of TNF-alpha and give some rationale for use of TNF-alpha antagonists in cancer prevention and treatment.

Discovery and mechanism of ustekinumab: a human monoclonal antibody targeting interleukin-12 and interleukin-23 for treatment of immune-mediated disorders.

Monoclonal antibody (mAb) therapy was first established upon the approval of a mouse antibody for treatment of human acute organ rejection. However, the high incidence of immune response against the mouse mAb restricted therapeutic utility. Development of chimeric, "humanized" and human mAbs broadened therapeutic application to immune-mediated diseases requiring long-term treatment. Indeed, mAb therapeutics targeting soluble cytokines are highly effective in numerous immune-mediated disorders. A recent example is ustekinumab, a first-in-class therapeutic human immunoglobulin G1 kappa mAb that binds to the interleukins (IL)-12 and IL-23, cytokines that modulate lymphocyte function, including T-helper (Th) 1 and Th17 cell subsets. Ustekinumab was generated via recombinant human IL-12 immunization of human immunoglobulin (hu-Ig) transgenic mice. Ustekinumab binds to the p40 subunit common to IL-12 and IL-23 and prevents their interaction with the IL-12 receptor ß1 subunit of the IL-12 and IL-23 receptor complexes. Ustekinumab is approved for treatment of moderate-to-severe plaque psoriasis and has demonstrated efficacy in Crohn disease and psoriatic arthritis. The clinical characterization of ustekinumab continues to clarify our understanding of human immune pathologies and may offer a novel therapeutic option for certain immune-mediated diseases.

Evaluation of an anti-tumor necrosis factor therapeutic in a mouse model of Niemann-Pick C liver disease.

BACKGROUND: Niemann-Pick type C (NPC) disease is a lysosomal storage disease characterized by the accumulation of cholesterol and glycosphingolipids. The majority of NPC patients die in their teen years due to progressive neurodegeneration; however, half of NPC patients also suffer from cholestasis, prolonged jaundice, and hepatosplenomegaly. We previously showed that a key mediator of NPC liver disease is tumor necrosis factor (TNF) α, which is involved in both proinflammatory and apoptotic signaling cascades. In this study, we tested the hypothesis that blocking TNF action with an anti-TNF monoclonal antibody (CNTO5048) will slow the progression of NPC liver disease. METHODOLOGY/PRINCIPAL FINDINGS: Treatment of wild-type C57BL/6 mice with NPC1-specific antisense oligonucleotides led to knockdown of NPC1 protein expression in the liver. This caused classical symptoms of NPC liver disease, including hepatic cholesterol accumulation, hepatomegaly, elevated serum liver enzymes, and lipid laden macrophage accumulation. In addition, there was a significant increase in the number of apoptotic cells and a proliferation of stellate cells. Concurrent treatment of NPC1 knockdown mice with anti-TNF had no effect on the primary lipid storage or accumulation of lipid-laden macrophages. However, anti-TNF treatment slightly blunted the increase in hepatic apoptosis and stellate cell activation that was seen with NPC1 knockdown. CONCLUSIONS/SIGNIFICANCE: Current therapeutic options for NPC disease are limited. Our results provide proof of principle that pharmacologically blocking the TNF-α inflammatory cascade can slightly reduce certain markers of NPC disease. Small molecule inhibitors of TNF that penetrate tissues and cross the blood-brain barrier may prove even more beneficial.

Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α.

We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018).  The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry.  In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration.  In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.

Tumor necrosis factor deficiency inhibits mammary tumorigenesis and a tumor necrosis factor neutralizing antibody decreases mammary tumor growth in neu/erbB2 transgenic mice.

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine that is synthesized and secreted by cells of the immune system, as well as by certain epithelia and stroma. Based on our previous studies demonstrating TNF-stimulated proliferation of normal and malignant mammary epithelial cells, we hypothesized that TNF might promote the growth of breast cancer in vivo. To test this, we generated bigenic mice that overexpressed activated neu/erbB2 in the mammary epithelium and whose TNF status was wild-type, heterozygous, or null. Mammary tumorigenesis was significantly decreased in TNF-/- mice (n = 30) compared with that in TNF+/+ mice (n = 27), with a palpable tumor incidence of 10.0% and 44.4%, and palpable tumors/mouse of 0.10 +/- 0.06 and 0.67 +/- 0.17, respectively. Tumorigenesis in the heterozygous group fell between that in the TNF+/+ and TNF-/- groups, but was not significantly different from either of the homozygous groups. The decreased tumor development in the TNF-/- mice was associated with a decreased proliferative index in the lobular and ductal mammary epithelium. To further investigate the role of TNF in breast cancer, mammary tumor-bearing mice whose tumors overexpressed wild-type neu/erbB2 were treated with a TNF-neutralizing antibody or a control antibody for 4 weeks (n = 20/group). Mammary tumor growth was significantly inhibited in mice treated with the anti-TNF antibody compared with the control antibody. Together, these data show a stimulatory role for TNF in the growth of breast tumors and suggest that TNF antagonists may be effective in a subset of patients with breast cancer.

TNF-alpha drives remodeling of blood vessels and lymphatics in sustained airway inflammation in mice.

Inflammation is associated with blood vessel and lymphatic vessel proliferation and remodeling. The microvasculature of the mouse trachea provides an ideal opportunity to study this process, as Mycoplasma pulmonis infection of mouse airways induces widespread and sustained vessel remodeling, including enlargement of capillaries into venules and lymphangiogenesis. Although the mediators responsible for these vascular changes in mice have not been identified, VEGF-A is known not to be involved. Here, we sought to determine whether TNF-alpha drives the changes in blood vessels and lymphatics in M. pulmonis-infected mice. The endothelial cells, but not pericytes, of blood vessels, but not lymphatics, were immunoreactive for TNF receptor 1 (TNF-R1) and lymphotoxin B receptors. Most TNF-R2 immunoreactivity was on leukocytes. Infection resulted in a large and sustained increase in TNF-alpha expression, as measured by real-time quantitative RT-PCR, and smaller increases in lymphotoxins and TNF receptors that preceded vessel remodeling. Substantially less vessel remodeling and lymphangiogenesis occurred when TNF-alpha signaling was inhibited by a blocking antibody or was silenced in Tnfr1-/- mice. When administered after infection was established, the TNF-alpha-specific antibody slowed but did not reverse blood vessel remodeling and lymphangiogenesis. The action of TNF-alpha on blood vessels is probably mediated through direct effects on endothelial cells, but its effects on lymphangiogenesis may require inflammatory mediators from recruited leukocytes. We conclude that TNF-alpha is a strong candidate for a mediator that drives blood vessel remodeling and lymphangiogenesis in inflammation.

Elucidating bone marrow edema and myelopoiesis in murine arthritis using contrast-enhanced magnetic resonance imaging.

OBJECTIVE: While bone marrow edema (BME) detected by magnetic resonance imaging (MRI) is a biomarker of arthritis, its nature remains poorly understood due to the limitations of clinical studies. In this study, MRI of murine arthritis was used to elucidate its cellular composition and vascular involvement. METHODS: BME was quantified using normalized bone marrow intensity (NBMI) from precontrast MRI and normalized marrow contrast enhancement (NMCE) following intravenous administration of gadopentate dimeglumine. Wild-type (WT) and tumor necrosis factor (TNF)-transgenic mice were scanned from 2 to 5 months of age, followed by histologic or fluorescence-activated cell sorting (FACS) analysis of marrow. In efficacy studies, TNF-transgenic mice were treated with anti-TNF or placebo for 8 weeks, and then were studied using bimonthly MRI and histologic analysis. RESULTS: NBMI values were similar in WT and TNF-transgenic mice at 2 months. The values in WT mice steadily decreased thereafter, with mean values becoming significantly different from those of TNF-transgenic mice at 3.5 months (mean +/- SD 0.29 +/- 0.08 versus 0.46 +/- 0.13; P < 0.05). Red to yellow marrow transformation occurred in WT but not TNF-transgenic mice, as observed histologically at 5 months. The marrow of TNF-transgenic mice that received anti-TNF therapy converted to yellow marrow, with lower NBMI values versus placebo at 6 weeks (mean +/- SD 0.26 +/- 0.07 versus 0.61 +/- 0.22; P < 0.05). FACS analysis of bone marrow revealed a significant correlation between NBMI values and CD11b+ monocytes (R2 = 0.91, P = 0.0028). Thresholds for "normal" red marrow versus pathologic BME were established, and it was also found that inflammatory marrow is highly permeable to contrast agent. CONCLUSION: BME signals in TNF-transgenic mice are caused by yellow to red marrow conversion, with increased myelopoiesis and increased marrow permeability. The factors that mediate these changes warrant further investigation.

Longitudinal assessment of synovial, lymph node, and bone volumes in inflammatory arthritis in mice by in vivo magnetic resonance imaging and microfocal computed tomography.

OBJECTIVE: To develop longitudinal 3-dimensional (3-D) measures of outcomes of inflammation and bone erosion in murine arthritis using contrast-enhanced magnetic resonance imaging (CE-MRI) and in vivo microfocal computed tomography (micro-CT) and, in a pilot study, to determine the value of entry criteria based on age versus synovial volume in therapeutic intervention studies. METHODS: CE-MRI and in vivo micro-CT were performed on tumor necrosis factor-transgenic (TNF-Tg) mice and their wild-type littermates to quantify the synovial and popliteal lymph node volumes and the patella and talus bone volumes, respectively, which were validated histologically. These longitudinal outcome measures were used to assess the natural history of erosive inflammatory arthritis. We also performed anti-TNF versus placebo efficacy studies in TNF-Tg mice in which treatment was initiated according to either age (4-5 months) or synovial volume (3 mm(3) as detected by CE-MRI). Linear regression was performed to analyze the correlation between synovitis and focal erosion. RESULTS: CE-MRI demonstrated the highly variable nature of TNF-induced joint inflammation. Initiation of treatment by synovial volume produced significantly larger treatment effects on the synovial volume (P = 0.04) and the lymph node volume (P < 0.01) than did initiation by age. By correlating the MRI and micro-CT data, we were able to demonstrate a significant relationship between changes in synovial and patellar volumes (R(2) = 0.75, P < 0.01). CONCLUSION: In vivo CE-MRI and micro-CT 3-D outcome measures are powerful tools that accurately demonstrate the progression of erosive inflammatory arthritis in mice. These methods can be used to identify mice with arthritis of similar severity before intervention studies are initiated, thus minimizing heterogeneity in outcome studies of chronic arthritis seen between genetically identical littermates.

Induction of dendritic cell maturation by IL-18.

IL-18 is a pluripotent proinflammatory cytokine produced primarily by antigen presenting cells involved in numerous aspects of immune regulation most notably on lymphoid cells. The effect of IL-18 stimulation on cells in the myeloid compartment, however, has been poorly studied. Human monocytes did not respond to IL-18. However, the human myelomonocytic cell line KG-1 and monocyte-derived dendritic cells (generated by GM-CSF+IL-4) showed a marked increase in CD83, HLA-DR, and several costimulatory molecules upon stimulation with IL-18. Furthermore, IL-18 decreased pinocytosis of these cells and increased their ability to stimulate alloreactive T cell proliferation, all characteristics of mature dendritic cells. These results suggest that IL-18 is involved in the maturation of myeloid DCs, but not differentiation of monocytes into DCs. The finding that IL-18 is involved in the maturation of dendritic cells is both novel and unexpected and indicates another important role for IL-18 as a key regulator of immune responses.

Anti-TNF-alpha antibody allows healing of joint damage in polyarthritic transgenic mice.

Anti-tumor-necrosis-factor-alpha (TNF-alpha) monoclonal antibody was used to treat Tg197 transgenic mice, which constitutively produce human TNF-alpha (hTNF-alpha) and develop a progressive polyarthritic disease. Treatment of both young (7- or 8-week-old) and aged (27- or 28-week-old) mice commenced when at least two limbs showed signs of moderate to severe arthritis. The therapeutic efficacy of anti-TNF-alpha antibody was assessed using various pathological indicators of disease progression. The clinical severity of arthritis in Tg197 mice was significantly reduced after anti-TNF-alpha treatment in comparison with saline-treated mice and in comparison with baseline assessments in both young and aged mice. The treatment with anti-TNF-alpha prevented loss of body weight. Inflammatory pathways as reflected by elevated circulating hTNF-alpha and local expression of various proinflammatory mediators were all diminished by anti-TNF-alpha treatment, confirming a critical role of hTNF-alpha in this model of progressive polyarthritis. More importantly, the amelioration of the disease was associated with reversal of existing structural damage, including synovitis and periosteal bone erosions evident on histology. Repair of cartilage was age dependent: reversal of cartilage degradation after anti-TNF-alpha treatment was observed in young mice but not in aged mice.