kn-93 and Lupus-Erythematosus--Systemic

We have found that calcium calmodulin kinase IV is increased in T cells, podocytes, and mesangial cells from patients with systemic lupus erythematosus, as well as in lupus-prone mice, podocytes of patients with focal segmental glomerulosclerosis, and in mice injected with doxorubicin. We showed that this accounts for aberrant T cell function and glomerular damage. Using nanoparticles (nlg) loaded with a small drug inhibitor of calcium calmodulin kinase IV and tagged with antibodies directed to CD4 we have been able to show inhibition of autoimmunity and lupus nephritis. Also, using nlg tagged with antibodies to nephrin, we showed suppression of nephritis in lupus-prone mice and of glomerular damage in mice exposed to doxorubicin. We propose the development of approaches to deliver drugs to cells in a targeted and precise manner.

Topics: Animals; Antibiotics, Antineoplastic; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 4; CD4 Antigens; Disease Models, Animal; DNA Methylation; Doxorubicin; Drug Delivery Systems; Glomerulosclerosis, Focal Segmental; Humans; Lupus Erythematosus, Systemic; Lupus Nephritis; Membrane Proteins; Mice; Mice, Inbred MRL lpr; Molecular Targeted Therapy; Nanoparticles; Protein Kinase Inhibitors; Sulfonamides; T-Lymphocytes; T-Lymphocytes, Regulatory; Th17 Cells

To clarify the significance of immunometabolism in systemic lupus erythematosus (SLE), and to determine the effect of calcium/calmodulin-dependent protein kinase 4 (CaMK4) on T cell metabolism.. Metabolomic profiling was performed using capillary electrophoresis mass spectrometry in naive T cells from MRL/lpr mice treated with anti-CD3/CD28 antibodies in the absence or presence of a CaMK4 inhibitor (KN-93). The expression of GLUT1 and CaMK4 in CD4+ T cells from healthy controls (n = 16), patients with inactive SLE (n = 13), and patients with active SLE (n = 14) was examined by flow cytometry and quantitative polymerase chain reaction. In vitro experiments were performed to determine the effect of KN-93 on the expression of GLUT1 during Th17 cell differentiation in T cells from patients with SLE.. CaMK4 inhibition significantly decreased the levels of glycolytic intermediates such as glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, pyruvate, and lactate (P < 0.05), whereas it did not affect the levels of the pentose phosphate pathway intermediates such as 6-phospho-d-gluconate, ribulose-5-phosphate, ribose-5-phosphate, and phosphoribosyl pyrophosphate. The expression levels of GLUT1 and CaMK4 in effector memory CD4+ T cells were significantly higher in patients with active SLE compared to healthy controls (P < 0.01 and P < 0.05, respectively) and patients with inactive SLE (P < 0.05 and P < 0.01, respectively). A functional analysis revealed that CaMK4 inhibition decreased the expression of GLUT1 during Th17 cell differentiation (P < 0.01), followed by a reduction of interleukin-17 (IL-17) production (P < 0.05).. The results of the study indicate that the activity of CaMK4 could be responsible for glycolysis, which contributes to the production of IL-17, and CaMK4 may contribute to aberrant expression of GLUT1 in T cells from patients with active SLE.

Topics: Adult; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Case-Control Studies; CD4-Positive T-Lymphocytes; Cell Differentiation; Female; Fructosediphosphates; Fructosephosphates; Glucose Transporter Type 1; Glucose-6-Phosphate; Glycolysis; Humans; Immunologic Memory; Interleukin-17; Lactic Acid; Lupus Erythematosus, Systemic; Male; Metabolome; Metabolomics; Mice; Mice, Inbred MRL lpr; Middle Aged; Pentose Phosphate Pathway; Protein Kinase Inhibitors; Pyruvic Acid; Sulfonamides; Th17 Cells

Treatment of autoimmune diseases is still largely based on the use of systemically acting immunosuppressive drugs, which invariably cause severe side effects. Calcium/calmodulin-dependent protein kinase IV is involved in the suppression of IL-2 and the production of IL-17. Its pharmacologic or genetic inhibition limits autoimmune disease in mice. In this study, we demonstrate that KN93, a small-molecule inhibitor of calcium/calmodulin-dependent protein kinase IV, targeted to CD4(+) T cells via a nanolipogel delivery system, markedly reduced experimental autoimmune encephalomyelitis and was 10-fold more potent than the free systemically delivered drug in the lupus mouse models. The targeted delivery of KN93 did not deplete T cells but effectively blocked Th17 cell differentiation and expansion as measured in the spinal cords and kidneys of mice developing experimental autoimmune encephalomyelitis or lupus, respectively. These results highlight the promise of cell-targeted inhibition of molecules involved in the pathogenesis of autoimmunity as a means of advancing the treatment of autoimmune diseases.

Topics: Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 4; CD4-Positive T-Lymphocytes; Cell Differentiation; Cells, Cultured; Encephalomyelitis, Autoimmune, Experimental; Humans; Immunosuppression Therapy; Lupus Erythematosus, Systemic; Mice; Mice, Inbred C57BL; Mice, Inbred MRL lpr; Nanogels; Polyethylene Glycols; Polyethyleneimine; Sulfonamides; Th17 Cells

Foxp3(+) regulatory T cells (Treg) are pivotal for the maintenance of peripheral tolerance and prevent development of autoimmune diseases. We have reported that calcium/calmodulin-dependent protein kinase IV (CaMK4) deficient MRL/lpr mice display less disease activity by promoting IL-2 production and increasing the activity of Treg cells. To further define the mechanism of CaMK4 on Treg cells in systemic lupus erythematosus (SLE), we used the Foxp3-GFP reporter mice and treated them with KN-93, an inhibitor of CaMK4.. We generated MRL/lpr Foxp3-GFP mice to record Treg cells; stimulated naïve CD4(+) T cells from MRL/lpr Foxp3-GFP mice under Treg polarizing conditions in the absence or presence of KN-93; evaluated the number of GFP positive cells in lymphoid organs and examined skin and kidney pathology at 16 weeks of age. We also examined the infiltration of cells and recruitment of Treg cells in the kidney.. We show that culture of MRL/lpr Foxp3-GFP T cells in the presence of KN-93 promotes Treg differentiation in a dose-dependent manner. Treatment of MRL/lpr Foxp3-GFP mice with KN-93 results in a significant induction of Treg cells in the spleen, peripheral lymph nodes and peripheral blood and this is accompanied by decreased skin and kidney damage. Notably, KN-93 clearly diminishes the accumulation of inflammatory cells along with reciprocally increased Treg cells in target organ.. Our results indicate that KN-93 treatment enhances the generation of Treg cells in vitro and in vivo highlighting its potential therapeutic use for the treatment of human autoimmune diseases.

Topics: Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Cell Count; Forkhead Transcription Factors; Interleukin-2; Lupus Erythematosus, Systemic; Mice, Inbred MRL lpr; Protein Kinase Inhibitors; Recovery of Function; Sulfonamides; T-Lymphocytes, Regulatory

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease associated with aberrant immune cell function. Treatment involves the use of indiscriminate immunosuppression, which results in significant side effects. SLE T cells express high levels of calcium/calmodulin-dependent protein kinase type IV (CaMKIV), which translocates to the nucleus upon engagement of the T cell receptor-CD3 complex and accounts for abnormal T cell function. The purpose of this study was to determine whether inhibition of CaMKIV would improve disease pathology.. We treated MRL/lpr mice with KN-93, a CaMKIV inhibitor, starting at week 8 or week 12 of age and continuing through week 16 and evaluated skin lesions, proteinuria, kidney histopathology, proinflammatory cytokine production, and costimulatory molecule expression. We also determined the effect of silencing of CAMK4 on interferon-γ (IFNγ) expression by human SLE T cells.. CaMKIV inhibition in MRL/lpr mice resulted in significant suppression of nephritis and skin disease, decreased expression of the costimulatory molecules CD86 and CD80 on B cells, and suppression of IFNγ and tumor necrosis factor α production. In human SLE T cells, silencing of CAMK4 resulted in suppression of IFNγ production.. We conclude that suppression of CaMKIV mitigates disease development in lupus-prone mice by suppressing cytokine production and costimulatory molecule expression. Specific silencing of CAMK4 in human T cells results in similar suppression of IFNγ production. Our data justify the development of small-molecule CaMKIV inhibitors for the treatment of patients with SLE.

Topics: Animals; Autoimmunity; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Disease Models, Animal; Female; Gene Silencing; Humans; Interferon-gamma; Kidney; Lupus Erythematosus, Systemic; Male; Mice; Nephritis; Protein Kinase Inhibitors; RNA, Small Interfering; Skin; Sulfonamides; T-Lymphocytes; Transfection