Rahul D. Pawar

Inhibition of Toll-Like Receptor-7 (TLR-7) or TLR-7 plus TLR-9 Attenuates Glomerulonephritis and Lung Injury in Experimental Lupus

Small nuclear RNA and associated lupus autoantigens activate B cells and dendritic cells via Toll-like receptor-7 (TLR-7); therefore, TLR-7 may represent a potential therapeutic target in lupus. MRL lpr mice were administered an injection of either saline or synthetic oligodeoxynucleotides with immunoregulatory sequences (IRS) that specifically block signaling via TLR-7 (IRS 661) or via TLR-7 and TLR-9 (IRS 954, which uses a active sequence from IRS 661 along with a TLR-9 inhibitory sequence) from weeks 11 to 24 of age. IRS 661 and IRS 954 both significantly reduced the weight of spleen and lymph nodes as well as serum levels of TNF as compared with saline-treated MRL lpr mice. Only IRS 661 but not IRS 954 significantly reduced serum levels of IL-12p40, anti-dsDNA IgG(2a), IgG(2b), and anti-Smith IgG. Both IRS localized to the kidney after intraperitoneal injection and significantly improved the activity index and chronicity index for lupus nephritis in MRL lpr mice. This was associated with significant reduction of renal glomerular and interstitial macrophage infiltrates and the number of interstitial T cells. Autoimmune lung injury was also attenuated with IRS 661 and IRS 954. These data demonstrate that TLR-7 antagonism, initiated after the onset of autoimmunity, can prevent autoimmune kidney and lung injury in MRL lpr mice. Concomitant blockade of TLR-9 with IRS 954 neutralized the effect of TLR-7 blockade on dsDNA IgG(2a), dsDNA IgG(2b), and Smith antigen autoantibodies but had neither additive nor opposing effects on autoimmune lung and kidney injury. Hence, TLR-7 is proposed as a novel and potential therapeutic target in systemic lupus erythematosus.

Spiegelmer Inhibition of CCL2/MCP-1 Ameliorates Lupus Nephritis in MRL-(Fas)lpr Mice

The monocyte chemoattractant protein CCL2 is crucial for monocyte and T cell recruitment from the vascular to the extravascular compartment at sites of inflammation. CCL2 is expressed in human lupus nephritis and was shown to mediate experimental lupus; therefore, CCL2 antagonists may be beneficial for therapy. This study describes the l-enantiomeric RNA oligonucleotide mNOX-E36, a so-called Spiegelmer that binds murine CCL2 with high affinity and neutralizes its action in vitro and in vivo. The mirror image configuration of the Spiegelmer confers nuclease resistance and thus excellent biostability. mNOX-E36 does not induce type I IFN via Toll-like receptor-7 or cytosolic RNA receptors, as recently shown for certain synthetic D-RNA. Autoimmune-prone MRL(lpr/lpr) mice that were treated with a polyethylene glycol form of mNOX-E36 from weeks 14 to 24 of age showed prolonged survival associated with a robust improvement of lupus nephritis, peribronchial inflammation, and lupus-like inflammatory skin lesions. Thus, mNOX-E36-based inhibition of CCL2 represents a novel strategy for the treatment of autoimmune tissue injury, such as lupus nephritis.

Toll-Like Receptor-7 Modulates Immune Complex Glomerulonephritis

Viral infections may trigger immune complex glomerulonephritis via Toll-like receptors (TLR), as certain TLR trigger immunity upon recognition of viral nucleic acids. On the basis of previous findings regarding viral double-stranded RNA and TLR3 in experimental lupus erythematosus, a similar role for TLR7 that recognizes viral single-stranded RNA was hypothesized. Immunostaining of kidney sections of nephritic MRLlpr/lpr mice revealed TLR7 expression in infiltrating ER-HR3-positive macrophages and few CD11c-positive dendritic cells but not in glomerular mesangial cells as observed for TLR3. This finding was consistent with the distribution pattern of intravenously injected single-stranded RNA in nephritic MRLlpr/lpr mice. TLR7 ligation activated monocytes and dendritic cells, both isolated from MRLlpr/lpr mice, to secrete IFN-alpha, IL-12p70, IL-6, and CCL2. In vivo, a single injection of the TLR7 ligand imiquimod increased serum levels of IL-12p70, IFN-alpha, and IL-6. A course of 25 microg of imiquimod given every other day from week 16 to 18 of age aggravated lupus nephritis in MRLlpr/lpr mice. This was associated with increased glomerular immune complex deposits as well as interstitial expression of CCL2 in imiquimod-treated MRLlpr/lpr mice. Different types of viral nucleic acids seem to modulate systemic autoimmunity through specific interactions with their respective TLR. Different TLR expression profiles on immune cell subsets and nonimmune parenchymal cell types determine the molecular mechanisms involved in viral infection-associated exacerbation of lupus nephritis and possibly other types of immune complex glomerulonephritis.

Requirement of toll-like receptor 7 for pristane-induced production of autoantibodies and development of murine lupus nephritis†

OBJECTIVE The detection of high titers of antibodies against small nuclear ribonucleoproteins (snRNP) is a diagnostic finding in patients in whom systemic lupus erythematosus (SLE) is suspected. Endogenous RNA molecules within snRNP trigger Toll-like receptor 7 (TLR-7) activation in B cells and dendritic cells, leading to anti-snRNP antibody production, which is associated with the development of immune complex nephritis in SLE. The purpose of this study was to investigate the role of TLR-7 in anti-snRNP antibody production and renal disease in SLE induced by an exogenous factor in the absence of genetic predisposition, using the pristane-induced murine lupus model. METHODS Serum autoantibodies, IgG isotypes, and cytokine levels in pristane-treated wild-type and TLR-7-deficient mice were analyzed by enzyme-linked immunosorbent assay. Histopathologic changes in mouse kidneys were determined by light immunofluorescence microscopy. Cell subsets in splenocytes and peritoneal lavage cells from the mice were examined by flow cytometry. RESULTS We found that anti-snRNP antibody production induced by pristane treatment was entirely dependent on the expression of TLR-7, whereas anti-double-stranded DNA antibody production was not affected by a lack of TLR-7. Impaired anti-snRNP antibody production in TLR-7-deficient mice was paralleled by lower levels of glomerular IgG and complement deposits, as well as less severe glomerulonephritis. CONCLUSION TLR-7 is specifically required for the production of RNA-reactive autoantibodies and the development of glomerulonephritis in pristane-induced murine lupus, a model of environmentally triggered SLE in the absence of genetic susceptibility to autoimmunity. Specific interference with TLR-7 activation by endogenous TLR-7 ligands may therefore be a promising novel strategy for the treatment of SLE.

Ligands to Nucleic Acid–Specific Toll-Like Receptors and the Onset of Lupus Nephritis

Lupus nephritis develops from a combination of genetic and environmental factors such as microbial infection. A role for microbial nucleic acids (e.g., via nucleic acid-specific Toll-like receptors [TLR]) was hypothesized, in this context, because microbial nucleic acids can trigger multiple aspects of autoimmunity in vitro and in vivo. Eight-week-old MRL(lpr/lpr) and MRL wild-type mice received an injection of pI:C RNA (ligand to TLR-3), imiquimod (ligand to TLR-7), or CpG-DNA (ligand to TLR-9) on alternate days for 2 wk. Only CpG-DNA triggered the onset of lupus nephritis in MRL(lpr/lpr) mice, as defined by diffuse proliferative glomerulonephritis associated with glomerular IgG and complement C3 deposition, proteinuria, and glomerular macrophage infiltrates. None of the compounds caused DNA autoantibody production or glomerulonephritis in MRL wild-type mice. The role of CpG-DNA to trigger lupus nephritis in MRL(lpr/lpr) mice was found to relate to its potent immunostimulatory effects at multiple levels: B cell IL12p40 production, B cell proliferation, double-stranded DNA autoantibody secretion, and dendritic cell IFN-alpha production. The induction of lupus nephritis by CpG-DNA is motif specific and could be prevented by co-injection of inhibitory DNA. In summary, among the ligands tested, CpG-DNA triggers lupus nephritis in genetically predisposed hosts. These data support the concept that systemic lupus erythematosus is triggered by pathogens that release CG-rich DNA.

G-Rich DNA Suppresses Systemic Lupus

Whereas the role of immune complexes in mediating renal cell and immune cell activation is well established, the contribution of sequence-specific immunomodulatory actions of the chromatin part remains unclear. Toll-like receptor-9 (TLR-9) mediates immunostimulatory effects of unmethylated microbial CpG-DNA. It was hypothesized that hypomethylated CpG-DNA in vertebrates may have similar effects and may contribute to disease progression in lupus nephritis. A synthetic G-rich DNA, known to block CpG-DNA effects, was used in this study. In macrophages, G-rich DNA suppressed CpG-DNA-but not LPS-induced production of CCL5 in a dose-dependent manner. Injections of G-rich DNA suppressed lymphoproliferation induced by CpG-DNA injections in mice. In MRL(lpr/lpr) mice with lupus nephritis, labeled G-rich DNA co-localized to glomerular immune complexes and was taken up into endosomes of TLR-9-positive infiltrating macrophages. Eleven-week-old MRL(lpr/lpr) mice that received injections of either saline or G-rich DNA for 13 wk revealed decreased lymphoproliferation and less autoimmune tissue injury in lungs and kidneys as compared with saline-treated controls. G-rich DNA reduced the levels of serum dsDNA-specific IgG2a as well as the renal immune complex deposits. This was consistent with the blocking effect of G-rich DNA on CpG-DNA-induced proliferation of B cells that were isolated from MRL(lpr/lpr) mice. As oligodeoxyribonucleotide 2114-treated MRL(lpr/lpr) mice were not exposed to exogenous CpG-DNA, these effects should relate to a blockade of CpG motifs in endogenous DNA. It is concluded that adjuvant activity of self-DNA contributes to the pathogenesis of lupus nephritis. Modulating the CpG-DNA-TLR-9 pathway may offer new opportunities for the understanding and treatment of lupus.

Delayed Chemokine Receptor 1 Blockade Prolongs Survival in Collagen 4A3–Deficient Mice with Alport Disease

Human Alport disease is caused by a lack of the alpha3-, 4-, or 5-chain of type IV collagen (COL4A). Affected humans and COL4A3-deficient mice develop glomerulosclerosis and progressive renal fibrosis in the presence of interstitial macrophages, but their contribution to disease progression is under debate. This question was addressed by treating COL4A3-deficient mice with BX471, an antagonist of chemokine receptor 1 (CCR1) that is known to block interstitial leukocyte recruitment. Treatment with BX471 from weeks 6 to 10 of life improved survival of COL4A3-deficient mice, associated with less interstitial macrophages, apoptotic tubular epithelial cells, tubular atrophy, interstitial fibrosis, and less globally sclerotic glomeruli. BX471 reduced total renal Cll5 mRNA expression by reducing the number of interstitial CCL5-positive cells in inflammatory cell infiltrates. Intravital microscopy of the cremaster muscle in male mice identified that BX471 or lack of CCR1 impaired leukocyte adhesion to activated vascular endothelium and transendothelial leukocyte migration, whereas leukocyte rolling and interstitial migration were not affected. Furthermore, in activated murine macrophages, BX471 completely blocked CCL3-induced CCL5 production. Thus, CCR1-mediated recruitment and local activation of macrophages contribute to disease progression in COL4A3-deficient mice. These data identify CCR1 as a potential therapeutic target for Alport disease or other progressive nephropathies associated with interstitial macrophage infiltrates.

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 MRLlpr/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 MRLlpr/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)‐γ 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 MRLlpr/lpr mice.

Viral 5′-triphosphate RNA and non-CpG DNA aggravate autoimmunity and lupus nephritis via distinct TLR-independent immune responses

Certain viral nucleic acids aggravate autoimmunity through nucleic acid‐specific TLR. Viral 5′‐triphosphate RNA (3P‐RNA) and double‐stranded non‐CpG DNA induce antiviral immunity via TLR‐independent pathways but their role in autoimmunity is unknown. Transient exposure of 16‐wk‐old MRLlpr/lpr mice to 3P‐RNA aggravated lupus nephritis by increasing IFN signaling and decreasing CD4+CD25+ T cells. By contrast, transient exposure to non‐CpG DNA exacerbate lupus nephritis in association with splenomegaly, lymphoproliferation, hypergammaglobulinaemia and increased B220+CD138+ plasma cells. Both, 3P‐RNA and non‐CpG DNA increased glomerular complement factor C3c deposits but both nucleic acid formats were less potent in aggravating renal pathology as compared with CpG DNA. 3P‐RNA and non‐CpG DNA also localized to the glomerular mesangial cells and activated cultured mesangial cells to produce IL‐6. We conclude, 3P‐RNA or non‐CpG DNA both trigger autoimmune disease in MRLlpr/lpr mice by specifically activating adaptive immunity but similarly enhance inflammation on the tissue level.

Anti-Ccl2 Spiegelmer permits 75% dose reduction of cyclophosphamide to control diffuse proliferative lupus nephritis and pneumonitis in MRL-Fas(lpr) mice

Cyclophosphamide (CYC) can control diffuse proliferative lupus nephritis (DPLN) by potent immunosuppression but remains associated with serious and life-threatening complications. Drugs that specifically target mediators of DPLN may help to reduce CYC dose and side effects. Monocyte chemoattractant protein (MCP-1)/CCL2 mediates monocyte and T cell recruitment in DPLN and Ccl2-specific l-enantiomeric RNA Spiegelmer mNOX-E36 neutralizes the biological effects of murine Ccl2 in vitro and in vivo. We injected MRLlpr/lpr mice with DPLN from 14 weeks of age with vehicle, weekly 30 mg/kg CYC (full dose), monthly 30 mg/kg CYC (one-fourth full dose), pegylated control Spiegelmer, pegylated anti-Ccl2 Spiegelmer (3/week), pegylated anti-Ccl2 Spiegelmer plus CYC one-fourth full dose and mycophenolate mofetil. At week 24, DPLN and autoimmune lung injury were virtually abolished with CYC full dose but not with CYC one-fourth full dose. The CYC one-fourth full dose/Spiegelmer combination was equipotent to CYC full dose on kidney and lung injury. CD3+CD4-CD8- and CD3+CD4+CD25+ T cells and serum interleukin-12p40 and tumor necrosis factor-α levels were all markedly affected by CYC full dose but not by CYC one-fourth full dose. No additive effects of anti-Ccl2 Spiegelmer were noted on bone marrow colony-forming unit-granulocyte macrophage counts and 7/4high monocyte counts, lymphoproliferation, and spleen T cell depletion. In summary, anti-Ccl2 Spiegelmer permits 75% dose reduction of CYC for controlling DPLN and pneumonitis in MRL-Fas(lpr) mice, sparing suppressive effects of full-dose CYC on myelosuppression and T cell depletion. We propose anti-Ccl2 Spiegelmer therapy as a novel strategy to reduce CYC toxicity in the treatment of severe lupus.