Lisa R. Schopf

A Selective Small Molecule IκB Kinase β Inhibitor Blocks Nuclear Factor κB-Mediated Inflammatory Responses in Human Fibroblast-Like Synoviocytes, Chondrocytes, and Mast Cells

IκB kinase (IKK) β is essential for inflammatory cytokine-induced activation of nuclear factor κB (NF-κB). NF-κB plays a pivotal role in the function of major cell types that contribute to the pathophysiological process of rheumatoid arthritis (RA). Here, we report the mechanism and the effect of the IKKβ inhibitor N-(6-chloro-7-methoxy-9H-β-carbolin-8-yl)-2-methylnicotinamide (ML120B), a β-carboline derivative, on NF-κB signaling and gene activation in RA-relevant cell systems. ML120B is a potent, selective, reversible, and ATP-competitive inhibitor of IKKβ with an IC50 of 60 nM when evaluated in an IκBα kinase complex assay. ML120B does not inhibit other IKK isoforms or a panel of other kinases. ML120B concentration-dependently inhibits tumor necrosis factor α (TNFα)-stimulated NF-κB signaling via inhibition of IκBα phosphorylation, degradation, and NF-κB translocation into the nucleus. For the first time, we have demonstrated that in human fibroblast-like synoviocytes, TNFα- or interleukin (IL)-1β-induced monocyte chemoattractant protein-1 regulated on activation, normal T cell expressed and secreted and production is IKKβ-dependent. In addition, for the first time, we have demonstrated that lipopolysaccharide- or peptidoglycan-induced cytokine production in human cord blood-derived mast cells is IKKβ-dependent. In addition, in human chondrocytes, ML120B inhibited IL-1β-induced matrix metalloproteinase production with an IC50 of approximately 1 μM. ML120B also blocked IL-1β-induced prostaglandin E2 production. In summary, ML120B blocked numerous NF-κB-regulated cell responses that are involved in inflammation and destructive processes in the RA joint. Our findings support the evaluation of IKKβ inhibitors as anti-inflammatory agents for the treatment of RA.

Mice deficient in PKC theta demonstrate impaired in vivo T cell activation and protection from T cell-mediated inflammatory diseases.

In the present study we have characterized T cell-driven immune function in mice that are genetically deficient in PKC theta. In response to simple immunologic stimulation invoked by in vivo T cell receptor (TCR) cross-linking, these mice showed significantly depressed plasma cytokine levels for IL-2, IL-4, IFNγ, and TNFα compared to wild-type (WT) mice. In parallel, spleen mRNA levels for these cytokines were reduced, and NF-κB activation was also reduced in PKC theta knockouts (KO). Injection of allogeneic cells into the footpad of PKC theta deficient mice provoked a significantly diminished local T cell response compared to WT mice similarly challenged. Unlike comparable cells from wild type mice, CD45RBhi T cells harvested from PKC theta deficient mice failed to induce colitis in the SCID-CD45RB cell transfer model of IBD. In another T cell-dependent model of inflammatory disease, PKC theta deficient animals developed far less severe neurologic signs and reduced spinal cord inflammatory cell infiltrate compared to WT controls in the MOG-induced EAE model. A fundamental role for PKC theta in T cell activation and in the development of T cell-mediated inflammatory diseases is indicated by these results.

Expression of Th2 cytokines and the stable Th2 marker ST2L in the absence of IL-4 during Leishmania major infection

In this study we characterized Th2 responses in the absence of IL‐4. We show that ST2L, a stable Th2 marker, is expressed at similar levels in Leishmania major‐infected IL‐4‐deficient (IL‐4– / –) and wild‐type BALB / c (IL‐4+ / +) mice. Th2 cytokines are secreted by in vivo differentiated lymphocytes in response to specific activation in the absence of IL‐4. Although IL‐13 is produced, its neutralization did not alter the outcome of infection. Thus, we demonstrate that Th2 differentiation as assessed by the expression of ST2L and the production of Th2 cytokines can occur in vivo in the absence of IL‐4.

Quantitative analysis of micro-CT imaging and histopathological signatures of experimental arthritis in rats.

Micro-computed tomographic (micro-CT) imaging provides a unique opportunity to capture 3-D architectural information in bone samples. In this study of pathological joint changes in a rat model of adjuvant-induced arthritis (AA), quantitative analysis of bone volume and roughness were performed by micro-CT imaging and compared with histopathology methods and paw swelling measurement. Micro-CT imaging of excised rat hind paws (n = 10) stored in formalin consisted of approximately 600 30-mum slices acquired on a 512 x 512 image matrix with isotropic resolution. Following imaging, the joints were scored from H&E stained sections for cartilage/bone erosion, pannus development, inflammation, and synovial hyperplasia. From micro-CT images, quantitative analysis of absolute bone volumes and bone roughness was performed. Bone erosion in the rat AA model is substantial, leading to a significant decline in tarsal volume (27%). The result of the custom bone roughness measurement indicated a 55% increase in surface roughness. Histological and paw volume analyses also demonstrated severe arthritic disease as compared to controls. Statistical analyses indicate correlations among bone volume, roughness, histology, and paw volume. These data demonstrate that the destructive progression of disease in a rat AA model can be quantified using 3-D micro-CT image analysis, which allows assessment of arthritic disease status and efficacy of experimental therapeutic agents.

T helper differentiation in resistant and susceptible B7-deficient mice infected with Leishmania major

The contribution of the costimulatory molecules B7–1 and B7–2 to the in vivo differentiation of Th cells remains controversial. The infection of resistant and susceptible strains of mice with the parasite Leishmania major provides a well‐established model for studying in vivo differentiation of CD4+ T cells. We have infected B7–1/B7‐2‐deficient mice onthe BALB/c and 129 background with L. major and subsequently examined different parameters of infection and cytokine responses upon restimulation of lymph node cells in vitro. BALB/c B7–2‐deficient and B7–1/B7–2‐double deficient mice are resistant to L. major, whereas BALB/c B7–1‐deficient mice remain as susceptible as wild‐type BALB/c mice. Differential expression of B7–1 and B7–2 can explain the distinct roles observed for these B7 costimulators in L. major infection.

Application of surface roughness analysis on micro-computed tomographic images of bone erosion: examples using a rodent model of rheumatoid arthritis.

Quantifying the bone erosion in preclinical models of rheumatoid arthritis is valuable for the evaluation of drug treatments. This study introduces a three-dimensional method for bone surface roughness measurement from micro–computed tomographic data obtained from rats subjected to collagen-induced arthritis (CIA), in which the degree of bone erosion is related to the severity and the duration of the disease. In two studies of rat CIA, the surface roughness of the talus bone following 21 days of disease increased 559% and 486% from the control group. At 41 days following disease induction, the roughness of the bone surface increased 857% above baseline. The roughness of the control samples was similar from each study (less than 4% different), demonstrating the robustness of the algorithm. Treatment with methotrexate at 0.1 mg/kg daily demonstrated significant protection from bone erosion, whereas the 0.05 mg/kg daily dose was not efficacious (98% versus 22% inhibition of roughness-measured bone erosion). The main advantage of such an algorithm is demonstrated in the preclinical drug study of rat CIA with methotrexate treatment, indicating the immediate utility of this approach in drug development studies.