David Shealy

Netting Neutrophils Induce Endothelial Damage, Infiltrate Tissues, and Expose Immunostimulatory Molecules in Systemic Lupus Erythematosus

An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these low-density granulocytes (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased levels of proinflammatory cytokines and type I IFNs. To directly establish LDGs as a distinct neutrophil subset, their gene array profiles were compared with those of autologous normal-density neutrophils and control neutrophils. LDGs significantly overexpress mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In contrast, gene profiles of lupus normal-density neutrophils do not differ from those of controls. LDGs have heightened capacity to synthesize neutrophils extracellular traps (NETs), which display increased externalization of bactericidal, immunostimulatory proteins, and autoantigens, including LL-37, IL-17, and dsDNA. Through NETosis, LDGs have increased capacity to kill endothelial cells and to stimulate IFN-α synthesis by plasmacytoid dendritic cells. Affected skin and kidneys from lupus patients are infiltrated by netting neutrophils, which expose LL-37 and dsDNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and its subsequent responses may play a prominent deleterious role.

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

Anti-tumor-necrosis-factor-α (TNF-α) monoclonal antibody was used to treat Tg197 transgenic mice, which constitutively produce human TNF-α (hTNF-α) 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-α antibody was assessed using various pathological indicators of disease progression. The clinical severity of arthritis in Tg197 mice was significantly reduced after anti-TNF-α treatment in comparison with saline-treated mice and in comparison with baseline assessments in both young and aged mice. The treatment with anti-TNF-α prevented loss of body weight. Inflammatory pathways as reflected by elevated circulating hTNF-α and local expression of various proinflammatory mediators were all diminished by anti-TNF-α treatment, confirming a critical role of hTNF-α 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-α treatment was observed in young mice but not in aged mice.

MRI and Quantification of Draining Lymph Node Function in Inflammatory Arthritis

Abstract:u2002 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 (R2= 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.

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

OBJECTIVEnWhile 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.nnnMETHODSnBME 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.nnnRESULTSnNBMI 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.nnnCONCLUSIONnBME 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.

Evaluation of anti-IL-6 monoclonal antibody therapy using murine type II collagen-induced arthritis.

Interleukin-6 is a multifunctional cytokine that is critical for T/B-cell differentiation and maturation, immunoglobulin secretion, acute-phase protein production, and macrophage/monocyte functions. Extensive research into the biology of IL-6 has implicated IL-6 in the pathophysiology and pathogenesis of RA. An anti-murine IL-6 mAb that neutralizes mouse IL-6 activities was tested in animal model of collagen-induced arthritis. Prophylactic treatment with anti-IL-6 mAb significantly reduced the incidence and severity of arthritis compared to control mAb treated mice. The mitogenic response of B and T cells isolated from the lymph nodes of anti-IL-6 treated mice was significantly reduced compared to cells isolated from control mAb treated mice. The overall histopathology score for paws from the anti-IL-6 treated mice was significantly reduced when compared to paws from mice treated with control mAb, including both inflammatory (synovitis and pannus) and erosive (erosions and architecture) parameters. Reduced loss of cartilage matrix components was also observed in the anti-IL-6 treated mice. Collectively, these data suggest that IL-6 plays a major role in the pathophysiology of rheumatoid arthritis, and thus support the potential benefit of anti-IL-6 mAb treatment in rheumatoid arthritis patients.

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.

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/β-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/β-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/β-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/β-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced β-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/β-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.

Divergent Gene Activation in Peripheral Blood and Tissues of Patients with Rheumatoid Arthritis, Psoriatic Arthritis and Psoriasis following Infliximab Therapy

Objective The immune inflammatory disorders rheumatoid arthritis (RA), psoriatic arthritis (PsA) and psoriasis (Ps) share common pathologic features and show responsiveness to anti-tumor necrosis factor (TNF) agents yet they are phenotypically distinct. The aim of this study was to examine if anti-TNF therapy is associated with divergent gene expression profiles in circulating cells and target tissues of patients with these diseases. Methods Peripheral blood CD14+ and CD14− cells were isolated from 9 RA, 12 PsA and 10 Ps patients before and after infliximab (IFX) treatment. Paired synovial (nu200a=u200a3, RA, PsA) and skin biopsies (nu200a=u200a5, Ps) were also collected. Gene expression was analyzed by microarrays. Results 26 out of 31 subjects responded to IFX. The transcriptional response of CD14+ cells to IFX was unique for the three diseases, with little overlap (<25%) in significantly changed gene lists (with PsA having the largest number of changed genes). In Ps, altered gene expression was more pronounced in lesional skin (relative to paired, healthy skin) compared to blood (relative to healthy controls). Marked suppression of up-regulated genes in affected skin was noted 2 weeks after therapy but the expression patterns differed from uninvolved skin. Divergent patterns of expression were noted between the blood cells and skin or synovial tissues in individual patients. Functions that promote cell differentiation, proliferation and apoptosis in all three diseases were enriched. RA was enriched in functions in CD14− cells, PsA in CD14+ cells and Ps in both CD14+ and CD14− cells, however, the specific functions showed little overlap in the 3 disorders. Conclusion Divergent patterns of altered gene expression are observed in RA, PsA and Ps patients in blood cells and target organs in IFX responders. Differential gene expression profiles in the blood do not correlate with those in target organs.

THU0042 Preclinical Characterization of Sirukumab, a Human Monoclonal Antibody that Targets Human Interleukin-6 Signaling

Background A significant fraction of patients with rheumatoid arthritis (RA) have an inadequate response to tumor necrosis factor α inhibitors as well as to biologics that target other pathways, such as anti-CD20 (rituximab), CTLA4-Ig (abatacept), or anti–interleukin-6 (IL-6) receptor (tocilizumab; TCZ). Due to the high concentrations of soluble IL-6 receptor (IL-6R) found in the blood and synovial fluid of patients with RA, we elected to examine whether targeting the cytokine IL-6 rather than IL-6R is a more efficient means to inhibit IL-6 signaling. Objectives To characterize sirukumab (SIR), a monoclonal human IgG1κ antibody specific for human IL-6, and compare neutralization of IL-6 signaling with TCZ, which targets the soluble and membrane forms of IL-6R. Methods Affinity and selectivity for human IL-6 were determined using kinetic exclusion, in vitro binding, and 7TD1 cell proliferation assays. The epitope recognized by SIR on human IL-6 was determined by protease protection and solution hydrogen-deuterium exchange, which was confirmed by alanine substitution. In vitro bioassays were used to demonstrate inhibition of IL-6 signaling and compare potency with TCZ. BALB/c mice challenged with human IL-6 produce serum amyloid A; this model was used to examine the potency of SIR in vivo. Results SIR bound to human IL-6 with high affinity (0.175 pM). Epitope mapping identified helix D, spatially proximal regions of helix A, and the loop before helix B as the binding sites for SIR on IL-6. These overlap with the site where soluble IL-6R binds to IL-6. SIR showed no binding of human IL-6 bound to the IL-6R/gp130 receptor complex on U937 cells. SIR did not recognize other ligands that signal through gp130, and species crossreactivity to IL-6 was limited to human and nonhuman primates. SIR, at doses of 5 and 0.5 mg/kg, significantly reduced expression of serum amyloid A in BALB/c mice challenged with human IL-6 compared to mice that received an isotype-matched, negative control antibody (P <0.05). In several bioassays utilizing U937, HepG2, and human endothelial cells, SIR ranged from 50-fold to 90-fold more potent than TCZ. Conclusions Current data suggests that SIR binds with high affinity and neutralizes the biological effects of human IL-6. In side-by-side bioassays, SIR appears to be more potent than TCZ. Phase 3 clinical trials are underway to establish the efficacy and safety of SIR in patients with RA. Acknowledgements Study sponsored by Janssen Research & Development, LLC, in collaboration with GlaxoSmithKline. Disclosure of Interest D. Gardner Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, E. Lacy Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, S. Wu Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, D. Shealy Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC

FRI0069 Neutralization of IL6 by Sirukumab (SIR) Inhibits Inflammation and Cellular Stress in a Human Vascular Surrogate System of Atherosclerosis

Background Rheumatoid arthritis (RA) and atherosclerosis are chronic inflammatory diseases that share pathologic and molecular features. RA doubles the risk of cardiovascular disease (CVD) compared to the non-RA population. Common RA treatments are anti-inflammatory by design, but effects on CVD are unclear. Previously we reported the effect of SIR on IL6 and tumor necrosis factor (TNF) α signaling using a human endothelial cell (EC) and smooth muscle cell (SMC) co-culture system. Here we report additional analyses further characterizing effects of SIR on atherosclerotic (ATH) and oxidative (OX) stress pathways. Objectives To examine if RA drugs decrease ATH signaling and cellular stress in vascular cells under CVD conditions. Methods An in vitro surrogate system that co-cultures human ECs and SMCs was used to assess effects of RA drugs on vascular cells. Fluid flow conditions that drive cardiovascular health and CVD were applied. Atheroprone flow conditions were based on human hemodynamics from the carotid bifurcation, a site prone to developing atherosclerosis. The culture medium contained atherogenic risk factors including in vivo concentrations of oxidized LDL (oxLDL), soluble IL6 receptor (sIL6R), and TNF. Using RNA sequencing and microarray, we performed transcriptomic and biologic pathway analyses of surrogate system response. We compared treatments targeting pathogenic RA pathways, including anti-IL6 (SIR), anti-IL6 receptor (tocilizumab; TCZ), anti-TNF (adalimumab; ADA), and a small molecule JAK inhibitor (tofacitinib; TOF). The magnitude of pathway response was calculated as the L2 norm of the log2 fold change of genes in the pathway. Results The combination of atheroprone flow, sIL6R, TNF, and oxLDL (RA-CVD conditions) induced a robust response of inflammatory and OX stress pathways compared to healthy conditions (atheroprone flow without TNF and with non-oxLDL and sIL6R). The anti-IL6/IL6R treatments (SIR, TCZ) improved RA-CVD conditions by inhibiting key pathogenic pathways, including ATH signaling and NRF2-mediated OX stress. SIR attenuated the magnitude of RA-CVD response in ATH and OX stress pathways vs IgG or vehicle control the most: by 17% (adj P=0.035; Wilcoxon signed-rank test) and 34% (adj P=0.094) in ECs; and 49% (adj P=1.4e-5) and 47% (adj P=1.1e-3) in SMCs, respectively. TCZ was similar to SIR in restoring ECs and SMCs to healthy conditions in both pathways; ADA showed a weaker, similar trend compared to IL6 inhibition. TOF was not effective in suppressing (and tended to exacerbate) ATH and OX stress pathways in ECs. Conclusions IL6 pathway inhibitors SIR and TCZ potently suppressed ATH and cellular stress in vitro. The degree of suppression suggests that these drugs may mitigate the effects of atherogenic factors sIL6R, TNF, and oxLDL. In contrast, TNF inhibitor ADA was a less effective inhibitor of key CVD pathways, while JAK inhibitor TOF tended to exacerbate CVD pathways. Collectively, the data suggest that IL6 inhibition may provide more CVD benefit compared to RA drugs targeting other pathways. Acknowledgements Study sponsored by Janssen Research & Development, LLC, in collaboration with GlaxoSmithKline. Disclosure of Interest R. Feaver Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, S. Collado Employee of: HemoShear, LLC, S. Hoang Employee of: HemoShear, LLC, E. Berzin Employee of: HemoShear, LLC, A. Armstrong Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, D. Gardner Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, H. Liu Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, A. Mackey Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, D. Manka Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, D. Shealy Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, B. Blackman Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC