Benzimidazole derivatives as tubulin polymerization inhibitors: Design, synthesis and in vitro cytotoxicity studies.

A new class of benzimidazole derivatives as tubulin polymerization inhibitors has been designed and synthesized in this study. The in vitro anticancer profile of the developed molecules was reconnoitred on selected human cancer cells. The highest cytotoxicity was illustrated by compounds 7n and 7u with IC50 values ranging from 2.55 to 17.89 µM with specificity toward SK-Mel-28 cells. They displayed 5-fold less cytotoxicity towards normal rat kidney epithelial NRK52E cells, which implies that they are not harmful to normal, healthy cells. The cellular staining procedures like AO/EB, DCFDA, and DAPI were applied to comprehend the inherent mechanism of apoptosis which displayed nuclear and morphological alterations. The Annexin V binding and JC-1 studies were executed to evaluate the extent of apoptosis and the decline in mitochondrial transmembrane potential in SK-Mel-28 cell lines. Compound 7n dose-dependently arrested the G2/M phase of the cell cycle and the target-based outcomes proposed tubulin polymerization inhibition by 7n (IC50 of 5.05±0.13 µM). Computational studies were also conducted on the tubulin protein (PDB ID: 3E22) to investigate the stabilized binding interactions of compounds 7n and 7u with tubulin, respectively.

Triazolo-linked benzimidazoles as tubulin polymerization inhibitors and DNA intercalators: Design, synthesis, cytotoxicity, and docking studies.

A simple "click" protocol was employed in the quest of synthesizing 1,2,3-triazole-linked benzimidazoles as promising anticancer agents on various human cancer cell lines such as A549, HCT116, SK-Mel-28, HT-29, and MCF-7. Compound 12j demonstrated significant cytotoxic potential towards SK-Mel-28 cancer cells (IC50 : 4.17 ± 0.09 µM) and displayed no cytotoxicity (IC50 : > 100 µM) against normal human BEAS-2B cells inferring its safety towards normal healthy cells. Further to comprehend the underlying apoptosis mechanisms, AO/EB, dichlorodihydrofluorescein diacetate (DCFDA), and 4',6-diamidino-2-phenylindole (DAPI) staining were performed, which revealed the nuclear and morphological alterations. Compound 12j displayed impairment in cellular migration and inhibited colony formation. The annexin V binding assay and JC-1 were implemented to evaluate the scope of apoptosis and the loss of the mitochondrial transmembrane potential in SK-Mel-28 cells. Cell-cycle analysis revealed that compound 12j arrested the cells at the G2/M phase in a dose-dependent manner. Target-based assays established the inhibition of tubulin polymerization by 12j at an IC50 value of 5.65 ± 0.05 µM and its effective binding with circulating tumor DNA as a DNA intercalator. The detailed binding interactions of 12j with tubulin and DNA were examined by docking studies on PDB ID: 3E22 and DNA hexamer (PDB ID: 1NAB), respectively.

New synthetic coumarinolignans as attenuators of pro-inflammatory cytokines in LPS-induced sepsis and carrageenan-induced paw oedema models.

OBJECTIVES: The aim of the study was to explore the inhibition efficacy of new synthetic coumarinolignans (SCLs) against the secretion of pro-inflammatory cytokines in two in vivo models of inflammation. METHODS: Four SCLs 1-4 were screened for their pro-inflammatory cytokine inhibitory potential through oral administration at a dose of 50 mg/kg body weight in lipopolysaccharide-induced mouse endotoxaemia and carrageenan-induced mouse paw oedema models. Levels of pro-inflammatory cytokines (IL-1ß, TNFα and IL-6) in blood and paw tissue samples were estimated using ELISA. Paw oedema was measured using a plethysmometer. Results were compared with a natural coumarinolignan, cleomiscosin A (5), and the structure-activity relationship (SAR) was interpreted. RESULTS AND DISCUSSION: Compound 2 had the greatest potential in the endotoxaemia model, exhibiting 66.41%, 62.56% and 43.15% inhibition of plasma IL-1ß, TNFα and IL-6 secretions, respectively. Further dose-dependent study revealed its anti-inflammatory potential even at dose of 10 mg/kg body weight with 24.42% decline in the level of IL-1ß. Nevertheless, SCLs 1, 3 and 4 showed marked inhibitory activity with 57.54%, 51.48% and 62.46% reduction in the levels of IL-1ß, respectively. Moreover, compound 2 decreased the plasma TNFα and IL-1ß levels to 50.03% and 36.58% along with the reduction of paw oedema volume in the local inflammation induced by carrageenan. All compounds including cleomiscosin A (5) were more effective against IL-1ß. By studying SAR, the presence of dihydroxyl groups in the phenyl ring of lignans was identified to be essential for the activity. Also, esterification of lignans and presence of a 4-methyl substituent in the coumarin nucleus were found to play some role in enhancing the activity. CONCLUSION: All four SCLs, especially compound 2, have shown vast potential to emerge out as promising anti-inflammatory drugs.

Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis.

Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-α-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-α/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure.

NLRP3 and ASC suppress lupus-like autoimmunity by driving the immunosuppressive effects of TGF-ß receptor signalling.

OBJECTIVES: The NLRP3/ASC inflammasome drives host defence and autoinflammatory disorders by activating caspase-1 to trigger the secretion of mature interleukin (IL)-1ß/IL-18, but its potential role in autoimmunity is speculative. METHODS: We generated and phenotyped Nlrp3-deficient, Asc-deficient, Il-1r-deficient and Il-18-deficient C57BL/6-lpr/lpr mice, the latter being a mild model of spontaneous lupus-like autoimmunity. RESULTS: While lack of IL-1R or IL-18 did not affect the C57BL/6-lpr/lpr phenotype, lack of NLRP3 or ASC triggered massive lymphoproliferation, lung T cell infiltrates and severe proliferative lupus nephritis within 6 months, which were all absent in age-matched C57BL/6-lpr/lpr controls. Lack of NLRP3 or ASC increased dendritic cell and macrophage activation, the expression of numerous proinflammatory mediators, lymphocyte necrosis and the expansion of most T cell and B cell subsets. In contrast, plasma cells and autoantibody production were hardly affected. This unexpected immunosuppressive effect of NLRP3 and ASC may relate to their known role in SMAD2/3 phosphorylation during tumour growth factor (TGF)-ß receptor signalling, for example, Nlrp3-deficiency and Asc-deficiency significantly suppressed the expression of numerous TGF-ß target genes in C57BL/6-lpr/lpr mice and partially recapitulated the known autoimmune phenotype of Tgf-ß1-deficient mice. CONCLUSIONS: These data identify a novel non-canonical immunoregulatory function of NLRP3 and ASC in autoimmunity.

Cathepsin S inhibition suppresses systemic lupus erythematosus and lupus nephritis because cathepsin S is essential for MHC class II-mediated CD4 T cell and B cell priming.

OBJECTIVES: Major histocompatibility complex (MHC) class II-mediated priming of T and B lymphocytes is a central element of autoimmunity in systemic lupus erythematosus (SLE) and lupus nephritis. The cysteine protease cathepsin S degrades the invariant peptide chain during MHC II assembly with antigenic peptide in antigen-presenting cells; therefore, we hypothesised that cathepsin S inhibition would be therapeutic in SLE. METHODS: We developed a highly specific small molecule, orally available, cathepsin S antagonist, RO5461111, with suitable pharmacodynamic and pharmacokinetic properties that efficiently suppressed antigen-specific T cell and B cell priming in vitro and in vivo. RESULTS: When given to MRL-Fas(lpr) mice with SLE and lupus nephritis, RO5461111 significantly reduced the activation of spleen dendritic cells and the subsequent expansion and activation of CD4 T cells and CD4/CD8 double-negative T cells. Cathepsin S inhibition impaired the spatial organisation of germinal centres, suppressed follicular B cell maturation to plasma cells and Ig class switch. This reversed hypergammaglobulinemia and significantly suppressed the plasma levels of numerous IgG (but not IgM) autoantibodies below baseline, including anti-dsDNA. This effect was associated with less glomerular IgG deposits, which protected kidneys from lupus nephritis. CONCLUSIONS: Together, cathepsin S promotes SLE by driving MHC class II-mediated T and B cell priming, germinal centre formation and B cell maturation towards plasma cells. These afferent immune pathways can be specifically reversed with the cathepsin S antagonist RO5461111, which prevents lupus nephritis progression even when given after disease onset. This novel therapeutic strategy could correct a common pathomechanism of SLE and other immune complex-related autoimmune diseases.

Podocyte loss involves MDM2-driven mitotic catastrophe.

Podocyte apoptosis as a pathway of podocyte loss is often suspected but rarely detected. To study podocyte apoptosis versus inflammatory forms of podocyte death in vivo, we targeted murine double minute (MDM)-2 for three reasons. First, MDM2 inhibits p53-dependent apoptosis; second, MDM2 facilitates NF-κB signalling; and third, podocytes show strong MDM2 expression. We hypothesized that blocking MDM2 during glomerular injury may trigger p53-mediated podocyte apoptosis, proteinuria, and glomerulosclerosis. Unexpectedly, MDM2 blockade in early adriamycin nephropathy of Balb/c mice had the opposite effect and reduced intra-renal cytokine and chemokine expression, glomerular macrophage and T-cell counts, and plasma creatinine and blood urea nitrogen levels. In cultured podocytes exposed to adriamycin, MDM2 blockade did not trigger podocyte death but induced G2/M arrest to prevent aberrant nuclear divisions and detachment of dying aneuploid podocytes, a feature of mitotic catastrophe in vitro and in vivo. Consistent with these observations, 12 of 164 consecutive human renal biopsies revealed features of podocyte mitotic catastrophe but only in glomerular disorders with proteinuria. Furthermore, delayed MDM2 blockade reduced plasma creatinine levels, blood urea nitrogen, tubular atrophy, interstitial leukocyte numbers, and cytokine expression as well as interstitial fibrosis. Together, MDM2-mediated mitotic catastrophe is a previously unrecognized variant of podocyte loss where MDM2 forces podocytes to complete the cell cycle, which in the absence of cytokinesis leads to podocyte aneuploidy, mitotic catastrophe, and loss by detachment. MDM2 blockade with nutlin-3a could be a novel therapeutic strategy to prevent renal inflammation, podocyte loss, glomerulosclerosis, proteinuria, and progressive kidney disease.

Endogenous and exogenous pentraxin-3 limits postischemic acute and chronic kidney injury.

Ischemia-reperfusion activates innate immunity and sterile inflammation, resulting in acute kidney injury. Since pentraxin 3 (PTX3) regulates multiple aspects of innate immunity and tissue inflammation, we tested whether PTX3 would be involved in renal ischemia-reperfusion injury. Renal pedicle clamping increased PTX3 serum levels, as well as PTX3 expression, inside the kidney but predominantly in CD45/CD11c(+) cells, a subpopulation of intrarenal mononuclear phagocytes. Lack of PTX3 aggravated postischemic acute kidney injury as evidenced by massive tubular necrosis, and TNF and IL-6 release, as well as massively increased neutrophil and macrophage infiltrates at 24 h. This was followed by tubular atrophy, interstitial fibrosis, and kidney shrinking 10 weeks later. In vivo microscopy uncovered increased leukocyte adhesion and transmigration in postischemic microvessels of Ptx3-deficient mice. Furthermore, injection of recombinant PTX3 up to 6 h after reperfusion prevented renal leukocyte recruitment and postischemic kidney injury. Thus, local PTX3 release from a subpopulation of intrarenal mononuclear phagocytes or delayed PTX3 treatment limits postischemic renal inflammation. Conversely, Ptx3 loss-of-function mutations predispose to postischemic acute kidney injury and subsequent chronic kidney disease.

Recombinant chaperonin 10 suppresses cutaneous lupus and lupus nephritis in MRL-(Fas)lpr mice.

BACKGROUND: Systemic lupus erythematosus (SLE) is still treated with global immunosuppressants with serious toxicities. We hypothesized that endogenous immunosuppressive molecules might be able to control SLE manifestations more specifically. Heat shock protein 10, or chaperonin 10 (Cpn10), is a secretory molecule that can suppress innate and adaptive immunity. METHODS: Recombinant human Cpn10 (100 µg per mouse) was given intraperitoneally to healthy-appearing female MRL-(Fas)lpr mice from 12 to 22 weeks of age. At the age of 22 weeks, mice were analysed for treatment outcome by harvesting organs, plasma and urine. RESULTS: Cpn10 entirely prevented cutaneous lupus lesions as compared to vehicle-treated mice. Cpn10 also suppressed lupus nephritis as evident from serum creatinine levels, albuminuria and the scores of disease activity and chronicity. Autoimmune lung disease was unaffected by Cpn10 treatment while overall survival of mice was prolonged. Cpn10 did not have any major effects on either dendritic cell or B-cell counts except T cells in spleen, plasma interferon-gamma, tumour necrosis factor-alpha, interleukin-10, anti-nuclear autoantibody levels or markers of lymphoproliferation. CONCLUSIONS: In summary, recombinant Cpn10 selectively prevents cutaneous lupus and suppresses nephritis in MRL-(Fas)lpr mice without affecting the underlying systemic autoimmune process. Hence, Cpn10 might be useful for the treatment of skin and kidney manifestations of SLE.

Mdm2 promotes systemic lupus erythematosus and lupus nephritis.

Systemic lupus erythematosus (SLE) is a polyclonal autoimmune syndrome directed against multiple nuclear autoantigens. Although RNA and DNA seem to have identical immunostimulatory effects on systemic and intrarenal inflammation, each seems to differ with regard to the propensity to induce mitogenic effects such as lymphoproliferation. To identify potential mechanisms by which DNA specifically contributes to the pathogenesis of lupus nephritis, we stimulated cells with immunostimulatory DNA or RNA in vitro and used microarray to compare the transcriptomes of RNA- and DNA-induced genes. Immunostimulatory DNA, but not RNA, induced Mdm2, which is a negative regulator of p53. In vivo, we observed greater expression and activation of Mdm2 in the spleen and kidneys in a mouse model of lupus (MRL-Fas(lpr) mice) than healthy controls. Treatment of MRL-Fas(lpr) mice with the Mdm2 inhibitor nutlin-3a prevented nephritis and lung disease and significantly prolonged survival. Inhibition of Mdm2 reduced systemic inflammation and abrogated immune complex disease by suppressing plasma cells and the production of lupus autoantibodies. In addition, nutlin-3a suppressed the abnormal expansion of all T cell subsets, including CD3(+)CD4(-)CD8(-) T cells, which associated with attenuated systemic inflammation. However, inhibiting Mdm2 did not cause myelosuppression or affect splenic regulatory T cells, neutrophils, dendritic cells, or monocytes. Taken together, these data suggest that the induction of Mdm2 promotes the expansion of plasma cells and CD3(+)CD4(-)CD8(-) T cells, which cause autoantibody production and immune complex disease in MRL-Fas(lpr) mice. Antagonizing Mdm2 may have therapeutic potential in lupus nephritis.

Interleukin-1 receptor-associated kinase-M suppresses systemic lupus erythematosus.

OBJECTIVES: Interleukin-1 receptor-associated kinase (IRAK)-M suppresses Toll-like receptor (TLR)-mediated activation of innate immunity during infection. A similar role was hypothesised for IRAK-M in autoimmunity. METHODS: Irak-m-deficient mice were crossed with autoimmune C57BL/6-lpr/lpr mice and detailed phenotype analysis was performed. RESULTS: Irak-m deficiency converted the mild autoimmune phenotype of C57BL/6-lpr/lpr mice into a massive lymphoproliferative syndrome with lethal autoimmune lung disease and lupus nephritis. Irak-m deficiency induced a number of interferon-related genes, cytokines and plasma cell survival factors in spleen cells of these mice. Irak-m-deficient C57BL/6-lpr/lpr mice showed expansion of autoreactive T cells, dysfunctional regulatory T cells and plasma cells which was associated with increased lupus autoantibody production. TLR7 antagonism almost completely abrogated this phenotype consistent with IRAK-M-mediated suppression of TLR7 signalling in vitro. CONCLUSIONS: These data identify a previously unknown function of IRAK-M-namely, suppression of TLR7-mediated autoimmunity-and mutant IRAK-M as a previously unknown genetic risk for murine SLE.

IRF4 deficiency abrogates lupus nephritis despite enhancing systemic cytokine production.

The IFN-regulatory factors IRF1, IRF3, IRF5, and IRF7 modulate processes involved in the pathogenesis of systemic lupus and lupus nephritis, but the contribution of IRF4, which has multiple roles in innate and adaptive immunity, is unknown. To determine a putative pathogenic role of IRF4 in lupus, we crossed Irf4-deficient mice with autoimmune C57BL/6-(Fas)lpr mice. IRF4 deficiency associated with increased activation of antigen-presenting cells in C57BL/6-(Fas)lpr mice, resulting in a massive increase in plasma levels of TNF and IL-12p40, suggesting that IRF4 suppresses cytokine release in these mice. Nevertheless, IRF4 deficiency completely protected these mice from glomerulonephritis and lung disease. The mice were hypogammaglobulinemic and lacked antinuclear and anti-dsDNA autoantibodies, revealing the requirement of IRF4 for the maturation of plasma cells. As a consequence, Irf4-deficient C57BL/6-(Fas)lpr mice neither developed immune complex disease nor glomerular activation of complement. In addition, lack of IRF4 impaired the maturation of Th17 effector T cells and reduced plasma levels of IL-17 and IL-21, which are cytokines known to contribute to autoimmune tissue injury. In summary, IRF4 deficiency enhances systemic inflammation and the activation of antigen-presenting cells but also prevents the maturation of plasma cells and effector T cells. Because these adaptive immune effectors are essential for the evolution of lupus nephritis, we conclude that IRF4 promotes the development of lupus nephritis despite suppressing antigen-presenting cells.

Dual blockade of the homeostatic chemokine CXCL12 and the proinflammatory chemokine CCL2 has additive protective effects on diabetic kidney disease.

Monocyte/ chemoattractant protein-1/chemokine ligand (CCL) 2 and stromal cell-derived factor-1/CXCL12 both contribute to glomerulosclerosis in mice with type 2 diabetes mellitus, through different mechanisms. CCL2 mediates macrophage-related inflammation, whereas CXCL12 contributes to podocyte loss. Therefore, we hypothesized that dual antagonism of these chemokines might have additive protective effects on the progression of diabetic nephropathy. We used chemokine antagonists based on structured l-enantiomeric RNA (so-called Spiegelmers) ie, the CCL2-specific mNOX-E36 and the CXCL12-specific NOX-A12. Male db/db mice, uninephrectomized at the age of 6 weeks, received injections of Spiegelmer, both Spiegelmers, nonfunctional control Spiegelmer, or vehicle from the age of 4 months for 8 weeks. Dual blockade was significantly more effective than monotherapy in preventing glomerulosclerosis. CCL2 blockade reduced glomerular leukocyte counts and renal-inducible nitric oxide synthase or IL-6 mRNA expression. CXCL12 blockade maintained podocyte numbers and renal nephrin and podocin mRNA expression. Consistently, CXCL12 blockade suppressed nephrin mRNA up-regulation in primary cultures of human glomerular progenitors induced to differentiate toward the podocyte lineage. All previously mentioned parameters were significantly improved in the dual-blockade group, which also suppressed proteinuria and was associated with the highest levels of glomerular filtration rate. Blood glucose levels and body weight were identical in all treatment groups. Dual chemokine blockade can have additive effects on the progression of diabetic kidney disease when the respective chemokine targets mediate different pathomechanisms of disease (ie, inflammation and progenitor differentiation toward the podocyte lineage).

An orally active chemokine receptor CCR2 antagonist prevents glomerulosclerosis and renal failure in type 2 diabetes.

The progression of diabetic nephropathy is associated with an infiltration of macrophages expressing different phenotypes. As classically activated chemokine receptor CCR2+ macrophages are thought to drive tissue inflammation and remodeling, we tested whether blocking CCR2 could reduce intrarenal inflammation and prevent glomerulosclerosis in type 2 diabetes. This was achieved with RO5234444, an orally active small-molecule CCR2 antagonist that blocks ligand binding, its internalization, and monocyte chemotaxis. Male type 2 diabetic db/db mice were uninephrectomized to increase glomerular hyperfiltration to accelerate the development of glomerulosclerosis. From 16 weeks until killing at 24 weeks of age, mice were chow fed with or without admixed antagonist to achieve a trough plasma concentration above IC50 for binding in the mouse. CCR2 blockade reduced circulating monocyte levels, but did not affect total leukocyte or neutrophil numbers, and was associated with a reduction in the number of macrophages and apoptotic podocytes in the glomerulus. This treatment resulted in a higher total number of podocytes, less glomerulosclerosis, reduced albuminuria, and a significantly improved glomerular filtration rate. This successful pre-clinical trial suggests that this antagonist may now be ready for testing in humans with the nephropathy of diabetes mellitus.

Bacterial CpG-DNA accelerates Alport glomerulosclerosis by inducing an M1 macrophage phenotype and tumor necrosis factor-α-mediated podocyte loss.

Loss of function mutations in the α3 or α4 chain of type IV collagen cause Alport nephropathy, characterized by progressive glomerulosclerosis. While studying the mechanisms that determine disease progression, we found that the evolution of kidney disease in Col4a3-deficient mice was associated with an influx of immune cell subsets including nonactivated macrophages. This suggested that intrarenal inflammation might accelerate Alport nephropathy. A possible mechanism might be the well-known enhancement of immune recognition by bacterial products. We found that exposure to bacterial endotoxin from 4 to 6 weeks of age did not affect disease progression, whereas an equipotent dose of cytosine-guanine (CpG)-DNA, a synthetic mimic of bacterial DNA, accelerated all aspects of Alport nephropathy and reduced the overall lifespan of Col4a3-deficient mice. This effect was associated with a significant increase of renal CD11b+/Ly6C(hi) macrophages, intrarenal production of inducible nitric oxide synthase, tumor necrosis factor (TNF)-α, interleukin-12, and CXCL10, and loss of podocytes. TNF-α was essential for acceleration of Alport nephropathy, as etanercept (a soluble TNF-α receptor) entirely abrogated the CpG-DNA effect. Thus, systemic exposure to CpG-DNA induces classically activated (M1) macrophages that enhance intrarenal inflammation and disease progression. Hence, factors that modulate the phenotype of renal macrophages can affect the progression of Alport nephropathy and, potentially, other types of chronic kidney diseases.

4SC-101, a novel small molecule dihydroorotate dehydrogenase inhibitor, suppresses systemic lupus erythematosus in MRL-(Fas)lpr mice.

Immunosuppressive treatments of systemic lupus (SLE) remain associated with significant toxicities; hence, compounds with better toxicity profiles are needed. Dihydroorotate dehydrogenase (DHODH) inhibition with leflunomide has proven to be effective in autoimmune diseases including SLE, but leflunomide can cause a variety of side effects. We hypothesized that 4SC-101, a novel DHODH inhibitor with a more favorable toxicity profile, would be as effective as high-dose cyclophosphamide (CYC) in controlling experimental SLE of female MRL(Fas)lpr mice. Daily oral gavage of 30, 100, and 300 mg/kg 4SC-101 from 12 to 22 weeks of age was compared with either vehicle or CYC treatment (30 mg/kg/week, i.p.) in terms of efficacy and toxicity. Three hundred milligrams per kilogram 4SC-101 was as effective as CYC in depleting spleen autoreactive T cells, B cells, and plasma cells as well as the respective DNA and RNA serum autoantibodies. This was associated with a comparable amelioration of the renal, dermal, and pulmonary SLE manifestations of MRL(Fas)lpr mice. However, even the highest dose of 4SC-101 had no effect on bone marrow neutrophil counts, which were significantly reduced in CYC-treated mice. Together, the novel DHODH inhibitor 4SC-101 is as effective as high dose CYC in controlling SLE without causing myelosuppression. Hence, DHODH inhibition with 4SC-101 might be suitable to treat active SLE with fewer side effects than CYC.

Lack of SIGIRR/TIR8 aggravates hydrocarbon oil-induced lupus nephritis.

Multiple genetic factors contribute to the clinical variability of spontaneous systemic lupus erythematosus (SLE) but their role in drug-induced SLE remain largely unknown. Hydrocarbon oil-induced SLE depends on mesothelial cell apoptosis and Toll-like receptor (TLR)-7-mediated induction of type I interferons. Hence, we hypothesized that TIR8/SIGIRR, an endogenous TLR inhibitor, prevents oil-induced SLE. Sigirr-deficient dendritic cells expressed higher TLR7 mRNA levels and TLR7 activation resulted in increased IL-12 production in vitro. In vivo, lack of SIGIRR increased surface CD40 expression on spleen CD11c(+) dendritic cells and MX-1, TNF, IL-12, BAFF and BCL-2 mRNA expression 6 months after pristane injection. Spleen cell counts of CD4(-)/CD8(-) 'autoreactive' T cells and B220(+) B cells were also increased in Sigirr(-/-) mice. Serum autoantibody analysis revealed that Sigirr deficiency specifically enhanced the production of rheumatoid factor (from 4 months of age) and anti-snRNP IgG (from 5 months of age), while anti-Smith IgG or anti-dsDNA IgG were independent of the Sigirr genotype. This effect was sufficient to significantly aggravate lupus nephritis in Sigirr-deficient mice. Structure model prediction identified the BB loop of SIGIRR's intracellular TIR domain to interact with TLR7 and MyD88. BB loop deletion was sufficient to completely abrogate SIGIRR's inhibitory effect on TLR7 signalling. Thus, TIR8/SIGIRR protects from hydrocarbon oil-induced lupus by suppressing the TLR7-mediated activation of dendritic cells, via its intracellular BB loop.

Viral RNA induces type I interferon-dependent cytokine release and cell death in mesangial cells via melanoma-differentiation-associated gene-5: Implications for viral infection-associated glomerulonephritis.

Viral RNA can trigger interferon signaling in dendritic cells via the innate recognition receptors melanoma-differentiation-associated gene (MDA)-5 and retinod-inducible gene (RIG)-I in the cytosol or via Toll-like receptors (TLRs) in intracellular endosomes. We hypothesized that viral RNA would also activate glomerular mesangial cells to produce type I interferon (IFN) via TLR-dependent and TLR-independent pathways. To test this hypothesis, we examined Toll/Interleukin-1 receptor domain-containing adaptor-inducing interferon-beta (TRIF)-deficient mice, which lack a key adaptor for TLR3 signaling. In primary mesangial cells, poly I:C RNA-mediated IFN-beta induction was partially TRIF dependent; however, when poly I:C RNA was complexed with cationic lipids to enhance cytosolic uptake, mesangial cells produced large amounts of IFN-alpha and IFN-beta independent of TRIF. Mesangial cells expressed RIG-I and MDA-5 and their mitochondrial adaptor IFN-beta promoter stimulator-1 as well, and small interfering RNA studies revealed that MDA5 but not RIG-I was required for cytosolic poly I:C RNA signaling. In addition, mesangial cells produced Il-6 on stimulation with IFN-alpha and IFN-beta, suggesting an autocrine proinflammatory effect. Indeed, blockade of IFN-alphabeta or lack of the IFNA receptor reduced viral RNA-induced Il-6 production and apoptotic cell death in mesangial cells. Furthermore, viral RNA/cationic lipid complexes increased focal necrosis in murine nephrotoxic serum nephritis in association with increased renal mRNA expression of IFN-related genes. Thus, TLR-independent recognition of viral RNA is a potent inducer of type I interferon in mesangial cells, which can be an important mediator of virally induced glomerulonephritis.

Trif is not required for immune complex glomerulonephritis: dying cells activate mesangial cells via Tlr2/Myd88 rather than Tlr3/Trif.

Viral RNA or bacterial products can activate glomerular mesangial cells via a subset of Toll-like receptors (Tlr). Because Tlr2-deficient mice were recently found to have attenuated nephrotoxic serum nephritis (NSN), we hypothesized that endogenous Tlr agonists can activate glomerular mesangial cells. Primary mesangial cells from C57BL/6 mice expressed Tlr1-6 and Tlr11 mRNA at considerable levels and produced Il-6 when being exposed to the respective Tlr ligands. Exposure to necrotic cells activated cultured primary mesangial cells to produce Il-6 in a Tlr2/Myd88-dependent manner. Apoptotic cells activated cultured mesangial cells only when being enriched to high numbers. Apoptotic cell-induced Il-6 release was Myd88 dependent, and only purified apoptotic cell RNA induced Trif signaling in mesangial cells. Does Trif signaling contribute to disease activity in glomerulonephritis? To answer this question, we induced autologous NSN by injection of NS raised in rabbits in Trif-mutant and wild-type mice. Lack of Trif did not alter the functional and histomorphological abnormalities of NSN, including the evolution of anti-rabbit IgG and anti-rabbit-specific nephritogenic T cells. We therefore conclude that apoptotic cell RNA is a poor activator of Trif signaling in mesangial cells and that necrotic cells' releases rather activate mesangial cells via the Tlr2/Myd88 signaling pathway.

Bacterial lipopeptide triggers massive albuminuria in murine lupus nephritis by activating Toll-like receptor 2 at the glomerular filtration barrier.

What are the molecular mechanisms of bacterial infections triggering or modulating lupus nephritis? In nephritic MRL(lpr/lpr) mice, transient exposure to bacterial cell wall components such as lipopeptide or lipopolysaccharide (LPS) increased splenomegaly, the production of DNA autoantibodies, and serum interleukin (IL)-6, IL-12 and tumour necrosis factor (TNF) levels, and aggravated lupus nephritis. Remarkably, bacterial lipopeptide induced massive albuminuria in nephritic but not in non-nephritic mice. This was associated with down-regulation of renal nephrin mRNA and redistribution from its normal localization at foot processes to the perinuclear podocyte area in nephritic MRL(lpr/lpr) mice. Bacterial lipopeptide activates Toll-like receptor 2 (TLR2), which we found to be expressed on cultured podocytes and glomerular endothelial cells. TNF and interferon (IFN)-gamma induced TLR2 mRNA and receptor expression in both cell types. Albumin permeability was significantly increased in cultured podocytes and glomerular endothelial cells upon stimulation by bacterial lipopeptide. LPS also induced moderate albuminuria. In summary, bacterial lipopeptide and LPS can aggravate glomerulonephritis but only lipopeptide potently induces severe albuminuria in MRL(lpr/lpr) mice.