A Phase 2 Randomized Controlled Study of Tralokinumab in Subjects with Idiopathic Pulmonary Fibrosis.

RATIONALE: IL-13 is a potential therapeutic target for idiopathic pulmonary fibrosis (IPF); preclinical data suggest a role in tissue fibrosis, and expression is increased in subjects with rapidly progressing disease. OBJECTIVES: Investigate efficacy and safety of tralokinumab, a human anti-IL-13 monoclonal antibody, in subjects with mild to moderate IPF. METHODS: Subjects received tralokinumab (400 or 800 mg), or placebo, intravenously every 4 weeks for 68 weeks. The primary endpoint was change from baseline to Week 52 in percent predicted FVC in the intention-to-treat population. Exploratory analyses included assessment of clinical response in subgroups with baseline serum periostin concentration above/below median. MEASUREMENTS AND MAIN RESULTS: The study was stopped due to lack of efficacy after interim analysis. Neither tralokinumab 400 mg nor tralokinumab 800 mg met the primary endpoint; least-squares mean difference (95% confidence interval) percent predicted FVC from baseline to Week 52: -1.77 (-4.13 to 0.59) (P = 0.140) and -1.41 (-3.73 to 0.91) (P = 0.234), respectively. The primary endpoint was also not met in either treatment group in subgroups defined by periostin baseline concentration. The percentage of subjects with decline in percent predicted FVC greater than or equal to 10% at Week 52 was numerically greater for tralokinumab-treated subjects compared with placebo. The most common treatment-emergent adverse events for tralokinumab 400 mg, tralokinumab 800 mg, and placebo were cough (17.5, 30.5, 22.8%), IPF progression and exacerbation (21.1, 16.9, 22.8%), and upper respiratory tract infection (17.5, 20.3, 12.3%), respectively. CONCLUSIONS: Tralokinumab demonstrated an acceptable safety and tolerability profile but did not achieve key efficacy endpoints. Clinical trial registered with www.clinicaltrials.gov (NCT01629667).

Role of the caspase-1 inflammasome in Salmonella typhimurium pathogenesis.

Caspase-1 is activated by a variety of stimuli after the assembly of the "inflammasome," an activating platform made up of a complex of the NOD-LRR family of proteins. Caspase-1 is required for the secretion of proinflammatory cytokines, such as interleukin (IL)-1beta and IL-18, and is involved in the control of many bacterial infections. Paradoxically, however, its absence has been reported to confer resistance to oral infection by Salmonella typhimurium. We show here that absence of caspase-1 or components of the inflammasome does not result in resistance to oral infection by S. typhimurium, but rather, leads to increased susceptibility to infection.

Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3.

Missense mutations in the CIAS1 gene cause three autoinflammatory disorders: familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal-onset multiple-system inflammatory disease. Cryopyrin (also called Nalp3), the product of CIAS1, is a member of the NOD-LRR protein family that has been linked to the activation of intracellular host defence signalling pathways. Cryopyrin forms a multi-protein complex termed 'the inflammasome', which contains the apoptosis-associated speck-like protein (ASC) and caspase-1, and promotes caspase-1 activation and processing of pro-interleukin (IL)-1beta (ref. 4). Here we show the effect of cryopyrin deficiency on inflammasome function and immune responses. Cryopyrin and ASC are essential for caspase-1 activation and IL-1beta and IL-18 production in response to bacterial RNA and the imidazoquinoline compounds R837 and R848. In contrast, secretion of tumour-necrosis factor-alpha and IL-6, as well as activation of NF-kappaB and mitogen-activated protein kinases (MAPKs) were unaffected by cryopyrin deficiency. Furthermore, we show that Toll-like receptors and cryopyrin control the secretion of IL-1beta and IL-18 through different intracellular pathways. These results reveal a critical role for cryopyrin in host defence through bacterial RNA-mediated activation of caspase-1, and provide insights regarding the pathogenesis of autoinflammatory syndromes.

Essential role for the C5a receptor in regulating the effector phase of synovial infiltration and joint destruction in experimental arthritis.

A characteristic feature of rheumatoid arthritis is the abundance of inflammatory cells in the diseased joint. Two major components of this infiltrate are neutrophils in the synovial fluid and macrophages in the synovial tissue. These cells produce cytokines including tumor necrosis factor alpha and other proinflammatory mediators that likely drive the disease through its effector phases. To investigate what mechanisms underlie the recruitment of these cells into the synovial fluid and tissue, we performed expression analyses of chemoattractant receptors in a related family that includes the anaphylatoxin receptors and the formyl-MetLeuPhe receptor. We then examined the effect of targeted disruption of two abundantly expressed chemoattractant receptors, the receptors for C3a and C5a, on arthritogenesis in a mouse model of disease. We report that genetic ablation of C5a receptor expression completely protects mice from arthritis.

Pharmacological targeting reveals distinct roles for CXCR2/CXCR1 and CCR2 in a mouse model of arthritis.

Neutrophils and monocytes are abundantly represented in the synovial fluid and tissue in rheumatoid arthritis patients. We therefore explored the effects of small molecule chemokine receptor antagonists to block migration of these cells in anti-collagen antibody-induced arthritis. Targeting neutrophil migration with the CXCR2/CXCR1 antagonist SCH563705 led to a dose-dependent decrease in clinical disease scores and paw thickness measurements and clearly reduced inflammation and bone and cartilage degradation based on histopathology and paw cytokine analyses. In contrast, targeting monocyte migration with the CCR2 antagonist MK0812 had no effect on arthritis disease severity. The pharmacodynamic activities of both SCH563705 and MK0812 were verified by assessing their effects on the peripheral blood monocyte and neutrophil populations. SCH563705 selectively reduced the peripheral blood neutrophil frequency, and caused an elevation in the CXCR2 ligand CXCL1. MK0812 selectively reduced the peripheral blood monocyte frequency, and caused an elevation in the CCR2 ligand CCL2. The much greater impact of CXCR2/CXCR1 antagonism relative to CCR2 antagonism in this model of arthritis highlights the therapeutic potential for targeting CXCR2/CXCR1 in human arthritides.

Safety, pharmacokinetics and pharmacodynamics of selgantolimod, an oral Toll-like receptor 8 agonist: a Phase Ia study in healthy subjects.

BACKGROUND: Selgantolimod is a novel oral, selective Toll-like receptor 8 (TLR8) agonist in development for the treatment of chronic hepatitis B (CHB). TLR8 is an endosomal innate immune receptor and a target for treatment of viral infections. This first-in-human study investigated the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of selgantolimod in healthy volunteers. METHODS: Of 71 subjects enrolled, 59 received a single dose of selgantolimod (0.5, 1.5, 3 or 5 mg) or placebo, and 12 were evaluated for food effect. Safety, PK and PD activity by induction of cytokines, chemokines and acute phase proteins were assessed. PK/PD analyses were conducted. RESULTS: Single doses of 0.5-5 mg were generally safe. No serious adverse events (AEs) or AEs leading to discontinuation were reported, and most were Grade 1 in severity. Selgantolimod displayed rapid absorption and dose-proportional PK and PD activity. Food had minimal effect on PK but resulted in diminished PD activity. In PK/PD analyses, near-saturation of induction for most evaluated biomarkers occurred at the 5-mg dose. CONCLUSIONS: Single doses of up to 5 mg selgantolimod were safe and induced dose-dependent PD responses. These data support evaluation of selgantolimod in combination with other agents in future clinical studies of CHB. Australian New Zealand Clinical Trials Registration: ACTRN12616001646437.

SLE Plasma Profiling Identifies Unique Signatures of Lupus Nephritis and Discoid Lupus.

Systemic lupus erythematosus (SLE) impacts multiple organ systems, although the causes of many individual SLE pathologies are poorly understood. This study was designed to elucidate organ-specific inflammation by identifying proteins that correlate with SLE organ involvement and to evaluate established biomarkers of disease activity across a diverse patient cohort. Plasma proteins and autoantibodies were measured across seven SLE manifestations. Comparative analyses between pathologies and correlation with the SLE Disease Activity Index (SLEDAI) were used to identify proteins associated with organ-specific and composite disease activity. Established biomarkers of composite disease activity, SLE-associated antibodies, type I interferon (IFN), and complement C3, correlated with composite SLEDAI, but did not significantly associate with many individual SLE pathologies. Two clusters of proteins were associated with renal disease in lupus nephritis samples. One cluster included markers of infiltrating leukocytes and the second cluster included markers of tissue remodelling. In patients with discoid lupus, a distinct signature consisting of elevated immunoglobulin A autoantibodies and interleukin-23 was observed. Our findings indicate that proteins from blood samples can be used to identify protein signatures that are distinct from established SLE biomarkers and SLEDAI and could be used to conveniently monitor multiple inflammatory pathways present in different organ systems.

MDP-induced interleukin-1beta processing requires Nod2 and CIAS1/NALP3.

Nucleotide-binding oligomerization domain (Nod)2 is a sensor of muramyl dipeptides (MDP) derived from bacterial peptidoglycan. Nod2 also plays a role in some autoinflammatory diseases. Cold-induced autoinflammatory syndrome 1 (CIAS1)/NACHT domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NALP3) has been suggested to be sufficient for MDP-dependent release of mature IL-1beta, but the role of Nod2 in this process is unclear. Using mice bearing selective gene deletions, we provide in vitro and in vivo data showing that MDP-induced IL-1beta release requires Nod2 and CIAS1/NALP3 as well as receptor-interacting protein-2 (Rip2), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. In contrast, MDP-dependent IL-6 production only requires Nod2 and Rip2. Together, our data provide a new understanding of this important pathway of IL-1beta production and allow for further studies of the role of these proteins within the broader context of inflammatory disease.

Development of a new ARCHITECT automated periostin immunoassay.

BACKGROUND: Periostin is being investigated as a potential biomarker for T-helper-2 (Th2)-driven asthma or eosinophilic inflammation and may help to identify patients more likely to benefit from interleukin-13-targeted treatments. We report the development and analytic performance of the investigational use only ARCHITECT Periostin Immunoassay, a new automated assay developed to detect serum periostin concentrations. METHODS: We assessed assay performance in terms of precision, sensitivity, linearity, interference from classical immunoassay interferents and representatives of common asthma medications, specimen handling, and isoform reactivity. The assay was also used to assess the biological variability of serum periostin concentrations in samples from healthy volunteers and from subjects with uncontrolled asthma (the intended use population). RESULTS: The percentage CVs for 5-day total precision, assessed using two instruments, was <6% across 2 controls and one serum-based panel. Limit of quantitation was 4ng/mL (dilution adjusted concentration), suiting the needs for this application. Dilution analysis yielded linear results and no endogenous sample or drug interferences were observed. All known periostin isoforms expressed in the mature human lung were detected by the assay. CONCLUSION: Our studies provide support that the ARCHITECT Periostin Immunoassay is a reliable and robust test for measuring serum periostin concentrations.

CCR2 and CXCR4 regulate peripheral blood monocyte pharmacodynamics and link to efficacy in experimental autoimmune encephalomyelitis.

BACKGROUND: CCR2 plays a key role in regulating monocyte trafficking to sites of inflammation and therefore has been the focus of much interest as a target for inflammatory disease. METHODS: Here we examined the effects of CCR2 blockade with a potent small molecule antagonist to determine the pharmacodynamic consequences on the peripheral blood monocyte compartment in the context of acute and chronic inflammatory processes. RESULTS: We demonstrate that CCR2 antagonism in vivo led to a rapid decrease in the number of circulating Ly6Chi monocytes and that this decrease was largely due to the CXCR4-dependent sequestration of these cells in the bone marrow, providing pharmacological evidence for a mechanism by which monocyte dynamics are regulated in vivo. CCR2 antagonism led to an accumulation of circulating CCL2 and CCL7 levels in the blood, indicating a role for CCR2 in regulating the levels of its ligands under homeostatic conditions. Finally, we show that the pharmacodynamic changes due to CCR2 antagonism were apparent after chronic dosing in mouse experimental autoimmune encephalomyelitis, a model in which CCR2 blockade demonstrated a dramatic reduction in disease severity, manifest in a reduced accumulation of monocytes and other cells in the CNS. CONCLUSION: CCR2 antagonism in vivo has tractable pharmacodynamic effects that can be used to align target engagement with biologic effects on disease activity.

NLRP3/cryopyrin is necessary for interleukin-1beta (IL-1beta) release in response to hyaluronan, an endogenous trigger of inflammation in response to injury.

Inflammation under sterile conditions is a key event in autoimmunity and following trauma. Hyaluronan, a glycosaminoglycan released from the extracellular matrix after injury, acts as an endogenous signal of trauma and can trigger chemokine release in injured tissue. Here, we investigated whether NLRP3/cryopyrin, a component of the inflammasome, participates in the inflammatory response to injury or the cytokine response to hyaluronan. Mice with a targeted deletion in cryopyrin showed a normal increase in Cxcl2 in response to sterile injuries but had decreased inflammation and release of interleukin-1beta (IL-1beta). Similarly, the addition of hyaluronan to macrophages derived from cryopyrin-deficient mice increased release of Cxcl2 but did not increase IL-1beta release. To define the mechanism of hyaluronan-mediated activation of cryopyrin, elements of the hyaluronan recognition process were studied in detail. IL-1beta release was inhibited in peritoneal macrophages derived from CD44-deficient mice, in an MH-S macrophage cell line treated with antibodies to CD44, or by inhibitors of lysosome function. The requirement for CD44 binding and hyaluronan internalization could be bypassed by intracellular administration of hyaluronan oligosaccharides (10-18-mer) in lipopolysaccharide-primed macrophages. Therefore, the action of CD44 and subsequent hyaluronan catabolism trigger the intracellular cryopyrin --> IL-1beta pathway. These findings support the hypothesis that hyaluronan works through IL-1beta and the cryopyrin system to signal sterile inflammation.

Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1beta in salmonella-infected macrophages.

Gram-negative bacteria that replicate in the cytosol of mammalian macrophages can activate a signaling pathway leading to caspase-1 cleavage and secretion of interleukin 1beta, a powerful host response factor. Ipaf, a cytosolic pattern-recognition receptor in the family of nucleotide-binding oligomerization domain-leucine-rich repeat proteins, is critical in such a response to salmonella infection, but the mechanism of how Ipaf is activated by the bacterium remains poorly understood. Here we demonstrate that salmonella strains either lacking flagellin or expressing mutant flagellin were deficient in activation of caspase-1 and in interleukin 1beta secretion, although transcription factor NF-kappaB-dependent production of interleukin 6 or the chemokine MCP-1 was unimpaired. Delivery of flagellin to the macrophage cytosol induced Ipaf-dependent activation of caspase-1 that was independent of Toll-like receptor 5, required for recognition of extracellular flagellin. In macrophages made tolerant by previous exposure to lipopolysaccharide, which abrogates activation of NF-kappaB and mitogen-activated protein kinases, salmonella infection still activated caspase-1. Thus, detection of flagellin through Ipaf induces caspase-1 activation independently of Toll-like receptor 5 in salmonella-infected and lipopolysaccharide-tolerized macrophages.

Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1.

Mutations in the NALP3/CIAS1/cryopyrin gene are linked to three autoinflammatory disorders: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and chronic infantile neurologic cutaneous and articular syndrome. NALP3, with the adaptor molecule ASC, has been proposed to form a caspase-1-activating "inflammasome," a complex with pro-IL1beta-processing activity. Here, we demonstrate the effect of NALP3 deficiency on caspase-1 function. NALP3 was essential for the ATP-driven activation of caspase-1 in lipopolysaccharide-stimulated macrophages and for the efficient secretion of the caspase-1-dependent cytokines IL-1alpha, IL-1beta, and IL-18. IL-1beta has been shown to play a key role in contact hypersensitivity; we show that ASC- and NALP3-deficient mice also demonstrate an impaired contact hypersensitivity response to the hapten trinitrophenylchloride. NALP3, however, was not required for caspase-1 activation by Salmonella typhimurium, and NALP3 deficiency only partially protects mice from the lethal effects of endotoxin. These data suggest that NALP3 plays a specific role in the caspase-1 activation pathway.

Distinct roles of TLR2 and the adaptor ASC in IL-1beta/IL-18 secretion in response to Listeria monocytogenes.

Apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) is an adaptor molecule that has recently been implicated in the activation of caspase-1. We have studied the role of ASC in the host defense against the intracellular pathogen Listeria monocytogenes. ASC was found to be essential for the secretion of IL-1beta/IL-18, but dispensable for IL-6, TNF-alpha, and IFN-beta production, in macrophages infected with Listeria. Activation of caspase-1 was abolished in ASC-deficient macrophages, whereas activation of NF-kappaB and p38 was unaffected. In contrast, secretion of IL-1beta, IL-6, and TNF-alpha was reduced in TLR2-deficient macrophages infected with Listeria; this was associated with impaired activation of NF-kappaB and p38, but normal caspase-1 processing. Analysis of Listeria mutants revealed that cytosolic invasion was required for ASC-dependent IL-1beta secretion, consistent with a critical role for cytosolic signaling in the activation of caspase-1. Secretion of IL-1beta in response to lipopeptide, a TLR2 agonist, was greatly reduced in ASC-null macrophages and was abolished in TLR2-deficient macrophages. These results demonstrate that TLR2 and ASC regulate the secretion of IL-1beta via distinct mechanisms in response to Listeria. ASC, but not TLR2, is required for caspase-1 activation independent of NF-kappaB in Listeria-infected macrophages.

CD1a-, b-, and c-restricted TCRs recognize both self and foreign antigens.

Individual CD1-restricted T cells can recognize either endogenous or foreign lipid Ags, but the extent to which the same CD1-restricted TCR can react to both self and microbial lipids is unknown. In this study, we have identified CD1a-, CD1b-, and CD1c-restricted T cells from normal human donors that induce cytolysis and secrete copious IFN-gamma in response to self-CD1 expressed on monocyte-derived dendritic cells. Remarkably, microbial Ags presented by CD1 are even more potent agonists for these same T cells. The alphabeta T cell receptors from such clones are diverse and confer specificity for both self-CD1 and foreign lipid Ags. The dual reactivity of these CD1-restricted cells suggests that the capacity for rapid responses to inflammatory stimuli without memory coexists with the capacity for strong Ag-specific responses and the generation of memory in vivo.

A mutant cell with a novel defect in MHC class I quality control.

COS7 (African Green Monkey kidney) cells stably transfected with the mouse MHC class I allele H-2K(b) were mutagenized, selected for low surface expression of endogenous MHC class I products, and subcloned. A mutant cell line, 4S8.12, expressing very low surface MHC class I (approximately 5% of parental levels) was identified. This cell line synthesized normal levels of the MHC class I H chain and beta(2)-microglobulin, as well as normal levels of TAP, tapasin, GRP78, calnexin, calreticulin, ERp57, and protein disulfide isomerase. Full-length OVA was processed to generate presented H-2K(b)-SIINFEKL complexes with equal efficiency in wild-type and mutant cells, demonstrating that proteasomes, as well as TAP and tapasin, functioned normally. Therefore, all the known components of the MHC class I Ag presentation pathway were intact. Nevertheless, primate (human and monkey) MHC class I H chain and beta(2)-microglobulin failed to associate to form the normal peptide-receptive complex. In contrast, mouse H chains associated with beta(2)-microglobulin normally and bound peptide at least as well as in wild-type cells. The 4S8.12 cells provide strong genetic evidence for a novel component in the MHC class I pathway. This as-yet unidentified gene is important in early assembly of primate, but not mouse, MHC class I complexes.

Informatics and the immune system: the expanding IL-1 and B7 protein families.

Remarkable advances in our knowledge of the genome sequence have led to the creation of databases that afford the opportunity to discover genes based on the presence of specific sequence motifs. The application of this strategy to the identification of proteins in families of immunological interest has had a visible impact on the sizes of the interleukin 1/18 and the CD28/B7 costimulatory molecule families, among many others. This accelerated pace of discovery presents a new challenge to match the rate of discovery with biological understanding of the functions of these extended protein families.

CD1-dependent dendritic cell instruction.

Both microbial products and T cell factors influence dendritic cell (DC) maturation. However, it is not known which T cells are capable of interacting with DCs at the initiation of adaptive immunity, when foreign antigen-specific T cells are rare. We show here that self-reactive CD1-restricted T cells can promote DC maturation by recognizing CD1 in the absence of foreign antigens. T cell recognition of all four CD1 isoforms can trigger DC maturation, but their distinct mechanisms of costimulation lead to profound differences in concomitant interleukin 12 p70 production. Distinct CD1-reactive T cells may thus differentially direct DC development early in the immune response, thereby controlling subsequent polarization of acquired immunity.

Differential dependence of CD4+CD25+ regulatory and natural killer-like T cells on signals leading to NF-kappaB activation.

Natural killer-like (NK) T, regulatory T (TR), and memory type T cells display surface phenotypes reminiscent of activated T cells. Previously, we reported that the generation of TR cells and, to a lesser extent, of memory type T cells, depends on IkappaB kinase 2. Here, we show that T cell-specific ablation of IkappaB kinase 2, in addition, completely precludes NKT cell development. T cell antigen receptor (TCR)-induced signals to activate NF-kappaB are essential for mature T cell activation, leading us to hypothesize that this pathway could play an important role in the generation of the antigen-driven T cell subsets comprising TR, memory type T, and NKT cells. TCR-mediated NF-kappaB activation critically depends on Bcl10 and PKCtheta. By using mice deficient for these proteins, we demonstrate that the generation of TR and, to a lesser extent, of memory type T cells, depends on Bcl10 and PKCtheta, and therefore, most likely on NF-kappaB activation initiated by TCR engagement. NKT cells, on the other hand, require PKCtheta for thymic development, whereas absence of Bcl10 leads primarily to the reduction of peripheral NKT cell numbers.

I kappa B kinase 2 deficiency in T cells leads to defects in priming, B cell help, germinal center reactions, and homeostatic expansion.

Signal transduction from proinflammatory stimuli leading to NF-kappa B-dependent gene expression is mediated by the I kappa B kinase 2 (IKK2/IKK beta). Therefore, IKK2 has become an important drug target for treatment of inflammatory conditions. T cells, whose activation depends to a large extent on the activity of NF-kappa B transcription factors, play important roles in inflammation and autoimmunity. Ablation of IKK2 specifically in T cells in CD4cre/Ikk2(FL) mice allows their survival and activation by polyclonal stimuli in vitro, suggesting that IKK2 is dispensable for T cell activation. We report in this study that IKK2-deficient T cells expand efficiently in response to superantigen administration in vivo, but are completely deficient in recall responses, most likely due to inefficient priming. IKK2-deficient T cells provide suboptimal B cell help and fail to support germinal center reactions. Finally, IKK2 is essential for homeostatic expansion of naive T cells, reflected by the inability of IKK2-deficient T cells to induce colitis in lymphopenic hosts.