A randomized, placebo-controlled, double-blind trial of canakinumab in children and young adults with sickle cell anemia.

Excessive intravascular release of lysed cellular contents from damaged red blood cells (RBCs) in patients with sickle cell anemia (SCA) can activate the inflammasome, a multiprotein oligomer promoting maturation and secretion of proinflammatory cytokines, including interleukin-1ß (IL-1ß). We hypothesized that IL-1ß blockade by canakinumab in patients with SCA would reduce markers of inflammation and clinical disease activity. In this randomized, double-blind, multicenter phase 2a study, patients aged 8 to 20 years with SCA (HbSS or HbSß0-thalassemia), history of acute pain episodes, and elevated high-sensitivity C-reactive protein >1.0 mg/L at screening were randomized 1:1 to received 6 monthly treatments with 300 mg subcutaneous canakinumab or placebo. Measured outcomes at baseline and weeks 4, 8, 12, 16, 20, and 24 included electronic patient-reported outcomes, hospitalization rate, and adverse events (AEs) and serious AEs (SAEs). All but 1 of the 49 enrolled patients were receiving stable background hydroxyurea therapy. Although the primary objective (prespecified reduction of pain) was not met, compared with patients in the placebo arm, patients treated with canakinumab had reductions in markers of inflammation, occurrence of SCA-related AEs and SAEs, and number and duration of hospitalizations as well as trends for improvement in pain intensity, fatigue, and absences from school or work. Post hoc analysis revealed treatment effects on weight, restricted to pediatric patients. Canakinumab was well tolerated with no treatment-related SAEs and no new safety signal. These findings demonstrate that the inflammation associated with SCA can be reduced by selective IL-1ß blockade by canakinumab with potential for therapeutic benefits. This trial was registered at www.clinicaltrials.gov as #NCT02961218.

The NLRP3 inflammasome pathway is activated in sarcoidosis and involved in granuloma formation.

Sarcoidosis is a disease characterised by granuloma formation. There is an unmet need for new treatment strategies beyond corticosteroids. The NLRP3 inflammasome pathway is expressed in innate immune cells and senses danger signals to elicit inflammatory interleukin (IL)-1ß; it has recently become a druggable target. This prompted us to test the role of the NLRP3 inflammasome and IL-1ß pathway in granuloma formation and sarcoidosis.19 sarcoid patients and 19 healthy volunteers were recruited into this pilot study. NLRP3 inflammasome activity was measured in bronchoalveolar lavage (BAL) cells and lung and skin biopsies using immunohistochemistry, Western blot, reverse-transcriptase PCR and ELISA. For in vivo experiments we used the trehalose 6,6'-dimycolate-granuloma mouse model and evaluated lung granuloma burden in miR-223 knockout and NLRP3 knockout mice, as well as the treatment effects of MCC950 and anti-IL-1ß antibody therapy.We found strong upregulation of the NLRP3 inflammasome pathway, evidenced by expression of activated NLRP3 inflammasome components, including cleaved caspase-1 and IL-1ß in lung granuloma, and increased IL-1ß release of BAL cells from sarcoid patients compared to healthy volunteers (p=0.006). mRNA levels of miR-223, a micro-RNA downregulating NLRP3, were decreased and NLRP3 mRNA correspondingly increased in alveolar macrophages from sarcoid patients (p<0.005). NLRP3 knockout mice showed decreased and miR-223 knockout mice increased granuloma formation compared to wild-type mice. Pharmacological interference using NLRP3 pathway inhibitor MCC950 or an anti-IL-1ß antibody resulted in reduced granuloma formation (p<0.02).In conclusion, our data provide evidence of upregulated inflammasome and IL-1ß pathway activation in sarcoidosis and suggest both as valid therapeutic targets.

Restraint stress fails to modulate cutaneous hypersensitivity responses in mice lacking the adenosine A1 receptor.

Psychological stress has long been associated with effects on immune function and disease. In particular, differential effects of acute and chronic stress on skin immunity occur in the rodent restraint stress model, with acute stress enhancing and chronic stress suppressing cutaneous hypersensitivity. Extracellular levels of adenosine are known to modulate diverse biological activities in the CNS and peripheral tissues and serve an important protective function against physiological stressors such as inflammation and ischemia. In this study, we utilized the restraint stress model and the skin sensitizer dinitrofluorobezene to test the hypothesis that perceived stress influences contact hypersensitivity through an adenosine A(1) receptor-mediated mechanism. We subjected hapten-sensitized A(1) receptor knockout (A1 KO) mice and their wild-type (WT) littermates to either acute (2.5 h) or chronic (5 h daily × 4 weeks) restraint stress, followed by hapten re-challenge of the pinna. Daily measurements of the resulting pinna swellings from each group were compared to reactions in non-stressed controls. In WT mice, pinna swelling was augmented in acutely stressed mice and suppressed in the chronically stressed group. In contrast, contact hypersensitivity responses in the A1 KO mice failed to be affected by either acute or chronic stress. Absence of the adenosine A(1) receptor did not affect levels of plasma corticosterone or urine catecholamines under these stressful conditions but did lead to reduced numbers of circulating neutrophil granulocytes compared to stressed WT animals. These results suggest that the adenosine A(1) receptor pathway plays a role in the process by which perceived psychological stress influences the contact hypersensitivity response.

CD4+CD25+ regulatory T cells suppress contact hypersensitivity reactions through a CD39, adenosine-dependent mechanism.

BACKGROUND: Injection of regulatory T (Treg) cells into sensitized mice abrogates the elicitation phase of contact hypersensitivity (CHS) reactions by blocking the adherence of leukocytes to vascular endothelium. OBJECTIVE: We set out to analyze whether adenosine, a suppressive factor recently described as produced by Treg cells, can account for the suppression of the effector T-cell-endothelial cell (EC) interaction. METHODS: T cells and ECs were cultured in the presence of adenosine, and expression of adhesion molecules and adhesion of T cells to ECs under shear stress were assessed. Furthermore, we injected Treg cells derived from ectonucleotidase-deficient (CD39-/-) mice into sensitized mice and analyzed the sticking and rolling of leukocytes during a CHS response using intravital microscopy. RESULTS: Adenosine or Treg cells, respectively, abrogated the adherence of effector T cells to ECs in vitro. Likewise, injection of adenosine and Treg cells abrogated the ear-swelling reaction, indicating a role of adenosine during Treg cell-induced suppression of CHS responses. As a source for Treg cell-derived adenosine, we identified the ectonucleotidase CD39 because CD39-deficient Treg cells did not prevent adhesion of leukocytes to the endothelium. Furthermore, we show that the impaired adhesion of effector T cells to inflamed endothelium was induced by adenosine-mediated downregulation of expression of E- and P-selectin on the vascular endothelium. CONCLUSION: Adenosine release by Treg cells is essential to block leukocyte adhesion to endothelium, providing a novel mechanism by which Treg cells mediate immune suppression in vivo.

Depletion of activated macrophages with a folate receptor-beta-specific antibody improves symptoms in mouse models of rheumatoid arthritis.

OBJECTIVES: Most therapies for autoimmune and inflammatory diseases either neutralize or suppress production of inflammatory cytokines produced by activated macrophages (e.g., TNFα, IL-1, IL-6, IL-17, GM-CSF). However, no approved therapies directly target this activated subset of macrophages. METHODS: First, we undertook to examine whether the folate receptor beta (FR-ß) positive subpopulation of macrophages, which marks the inflammatory subset in animal models of rheumatoid arthritis, might constitute the prominent population of macrophages in inflamed lesions in humans. Next, we utilized anti-FR-ß monoclonal antibodies capable of mediating antibody-dependent cell cytotoxicity (ADCC) to treat animal models of rheumatoid arthritis and peritonitis. RESULTS: Human tissue samples of rheumatoid arthritis, Crohn's disease, ulcerative colitis, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, chronic obstructive pulmonary disease, systemic lupus erythematosus, psoriasis, and scleroderma are all characterized by dramatic accumulation of macrophages that express FR-ß, a protein not expressed on resting macrophages or any other healthy tissues. A monoclonal antibody to FR-ß accumulates specifically in inflamed lesions of murine inflammatory disease models and successfully treats such models of rheumatoid arthritis and peritonitis. More importantly, elimination of FR-ß-positive macrophages upon treatment with an anti-FR-ß monoclonal antibody promotes the departure of other immune cells, including T cells, B cells, neutrophils, and dendritic cells from the inflamed lesions. CONCLUSIONS: These data suggest that specific elimination of FR-ß-expressing macrophages may constitute a highly specific therapy for multiple autoimmune and inflammatory diseases and that a recently developed human anti-human FR-ß monoclonal antibody (m909) might contribute to suppression of this subpopulation of macrophages.

The anergic state in sarcoidosis is associated with diminished dendritic cell function.

Sarcoidosis is a chronic inflammatory disease of unknown cause, characterized by granuloma formation similar to tuberculosis, but without clear evidence of a microbial infection. Because sarcoidosis is linked with clinical anergy and other evidence of diminished cellular immunity, we hypothesized that decreased skin delayed-type hypersensitivity (DTH) responses to recall Ags in affected individuals would be associated with decreased function of their blood dendritic cells (DCs). Our study involved ex vivo isolation, phenotyping, and functional testing of myeloid DCs (mDCs), plasmacytoid DCs, and T lymphocytes from blood of normal healthy volunteers and sarcoidosis subjects with active, untreated pulmonary disease. We found mDC function in the allogeneic MLR directly corresponded to the magnitude of skin DTH reactions to recall Ags in both sarcoidosis subjects and normal volunteers. However, both of these outcomes were significantly decreased in the sarcoidosis group. Diminished mDC function occurred despite up-regulated costimulatory and maturation markers. Clinical relevance is suggested by the inverse relationship between both mDC allogeneic responses and skin DTH responses with clinical disease severity as measured by chest radiograms. Because granulomas form when cellular immunity fails to clear antigenic stimuli, attenuated mDC function in sarcoidosis may contribute to susceptibility and persistence of the chronic inflammation characteristic of this disease.

Vanadate, an inhibitor of stromelysin and collagenase expression, suppresses collagen induced arthritis.

OBJECTIVE: Collagen induced arthritis (CIA) is a model of chronic inflammatory synovitis with pannus, neovascularization, and joint destruction similar to rheumatoid arthritis (RA). Matrix metalloproteinases (MMP) are involved in degradation of the extracellular matrix and joint destruction in RA. c-fos and c-jun are protooncogenes whose products combine to form activating protein (AP-1), a regulatory protein that is required for cell proliferation and the transcription of a variety of genes, including MMP such as collagenase and stromelysin. Administration of vanadium compounds suppresses c-fos/c-jun expression and AP-1 activity, resulting in inhibition of MMP expression in response to factors such as interleukin 1 (IL-1). We evaluated whether a vanadium AP-1 inhibitor could reduce MMP expression and subsequent joint damage in CIA. METHODS: Vanadate [bis (maltolato) oxovanadium (IV) (BMOV; 10 mg/kg/day)] and the reducing agent N-acetyl cysteine (NAC; 100 mg/kg/day) were given subcutaneously daily in an attempt to suppress established CIA in rats. NAC in combination with vanadate appeared to increase the efficacy of c-fos/c-jun inhibition, while decreasing toxicity. Controls were given NAC alone. Clinical, radiographic, and histologic measures were evaluated as well as synovial MMP and IL-1a expression. RESULTS: BMOV therapy, initiated on the day of onset of clinical arthritis, significantly reduced clinical arthritis within 2 days (p <0.05) compared to controls. Significance was maintained to the termination of the study on Day 18 post-arthritis onset (p < 0.005), with a maximum difference seen on Day 5 (p < 0.00001). Blinded radiographic scores at the completion of the protocols indicated less joint destruction in the experimental group compared to the control group (p < 0.005). Scanning and transmission electron microscopy confirmed the preservation of articular cartilage with therapy. In BMOV-treated rats, synovial mRNA expression of collagenase, stromelysin, and IL-la were reduced by 78%, 58%, and 85%, respectively, compared to controls. CONCLUSION: This is the first study of vanadate as a potential antirheumatic agent. Further study of this AP-1 and MMP inhibitor may lead to new treatment options in RA.

Nonpulmonary manifestations of sarcoidosis.

Sarcoidosis is one in a heterogeneous family of granulomatous disorders. The clinical manifestations of sarcoidosis can vary widely, depending on the patient and the tissues involved. Recent advances in our understanding of the immunologic steps leading to granuloma formation and persistence have yet to translate into more effective care for patients with this disease. This review discusses the immunology of granuloma formation and systemic disease, the various nonpulmonary expressions of the clinical disease, and the treatment options available to the practicing physician.

Thalidomide induces granuloma differentiation in sarcoid skin lesions associated with disease improvement.

Sarcoidosis, a chronic granulomatous disease of unknown etiology, is treated with immune suppressive drugs such as corticosteroids. Sarcoidosis patients have been reported to benefit clinically from treatment with thalidomide. We administered thalidomide for 16 weeks to eight patients with chronic skin sarcoidosis and evaluated the drug's effects before and with treatment. After thalidomide treatment, all skin biopsies showed decreases in granuloma size and reduction in epidermal thickness. We also observed extensive T cell recruitment into the granulomas, the appearance of multinucleated giant cells, and increased numbers of dermal Langerhans cells (CD1a(+)) and mature dendritic cells (CD83(+) or DC-LAMP(+)). Plasma IL-12 levels increased and remained elevated during the treatment period. We noted increased HLA-DR expression on peripheral blood lymphocytes and a corresponding drop in the naive T cell marker CD45RA. Our data suggest that thalidomide treatment of sarcoidosis results in granuloma differentiation to a Th1-type cellular immune response usually associated with protective immunity to tuberculosis and tuberculoid leprosy.