Hongyang Wang

P2X7 Blockade Attenuates Murine Lupus Nephritis by Inhibiting Activation of the NLRP3/ASC/Caspase 1 Pathway

OBJECTIVEnThe NLRP3 inflammasome plays key roles in inflammation and autoimmunity, and purinergic receptor P2X7 has been proposed to be upstream of NLRP3 activation. The aim of the present study, using murine models, was to investigate whether the P2X7 /NLRP3 inflammasome pathway contributes to the pathogenesis of lupus nephritis (LN).nnnMETHODSnMRL/lpr mice were treated with the selective P2X7 antagonist brilliant blue G (BBG) for 8 weeks. Following treatment, the severity of renal lesions, production of anti-double-stranded DNA (anti-dsDNA) antibodies, rate of survival, activation of the NLRP3/ASC/caspase 1 inflammasome pathway, and ratio of Th17 cells to Treg cells were evaluated. P2X7 -targeted small interfering RNA (siRNA) was also used for in vivo intervention. Similar evaluations were carried out in NZM2328 mice, a model of LN in which the disease was accelerated by administration of adenovirus-expressing interferon-α (AdIFNα).nnnRESULTSnSignificant up-regulation of P2X7 /NLRP3 inflammasome signaling molecules was detected in the kidneys of MLR/lpr mice as compared with normal control mice. Blockade of P2X7 activation by BBG suppressed NLRP3/ASC/caspase 1 assembly and the subsequent release of interleukin-1β (IL-1β), resulting in a significant reduction in the severity of nephritis and circulating anti-dsDNA antibodies. The lifespan of the treated mice was significantly prolonged. BBG treatment reduced the serum levels of IL-1β and IL-17 and the Th17:Treg cell ratio. Similar results were obtained by specific siRNA silencing of P2X7 in vivo. The effectiveness of BBG treatment in modulating LN was confirmed in NZM2328 mice with AdIFNα-accelerated disease.nnnCONCLUSIONnActivation of the P2X7 signaling pathway accelerates murine LN by activating the NLRP3/ASC/caspase 1 inflammasome, resulting in increased IL-1β production and enhanced Th17 cell polarization. Thus, targeting of the P2X7 /NLRP3 pathway should be considered as a novel therapeutic strategy in patients with lupus.

Role for Nephritogenic T Cells in Lupus Glomerulonephritis: Progression to Renal Failure Is Accompanied by T Cell Activation and Expansion in Regional Lymph Nodes

Autoreactive T cells are critical in the initiation and maintenance of autoantibody responses that are a hallmark of systemic lupus erythematosus. However, the direct contribution of T cells in end-organ disease like lupus glomerulonephritis (GN) is poorly understood. In this study, we investigated the role of T cells in progression of lupus GN in NZM2328 mice, a murine model of spontaneous systemic lupus erythematosus. At 26 wk of age, NZM2328 female mice showed glomerular immune complex deposits and acute proliferative GN. This was associated with up-regulation of MHC class II and the detection of T cells and CD11c+ dendritic cells in the glomeruli. The regional lymph nodes (LN) showed preferential activation of T cells and an oligoclonal T cell response with skewed expansion of certain Vβ families. This suggests an Ag-driven response occurring in the regional LN of nephritic mice during acute GN. In contrast, male NZM2328 mice developed glomerular immune complexes and acute GN, but rarely progressed to fatal chronic GN. Significantly, male kidneys at 40 wk of age did not have detectable dendritic cells and T cells in the glomeruli. Thus, glomerular immune complex deposition initiates an immune response against renal Ags in the regional LN, leading to T cell recruitment into the kidney during acute proliferative GN. This T cell activation and infiltration are influenced by gender-dependent end-organ factors and may determine the progression of acute GN to chronic GN and renal failure.

Cgnz1 allele confers kidney resistance to damage preventing progression of immune complex–mediated acute lupus glomerulonephritis

The mechanisms regulating acute and chronic glomerulonephritis are dependent on different genetic mechanisms, where the Cgnz1 allele confers kidney protection in immune complex–mediated proliferative lupus nephritis.

Interferon alpha on NZM2328.Lc1R27: Enhancing autoimmunity and immune complex-mediated glomerulonephritis without end stage renal failure

Interferon alpha (IFNα) may play a significant role in systemic lupus erythematosus (SLE) pathogenesis. Recent literature suggests that IFNα does not correlate with disease activities and blockade of IFNα is not effective in treating SLE. This study aims to delineate further the role of IFNα in SLE. 12-week old NZM2328 and its congenic NZM2328.Lc1R27 (R27) female mice were challenged with adenovirus-IFNα (adeno-IFNα) or adenovirus-LacZ (adeno-LacZ). Only adeno-IFNα treated NZM2328 developed severe proteinuria and died of chronic glomerulonephritis (GN) and end stage renal disease. Adeno-IFNα treated R27 did develop immune complex-mediated GN but had normal renal function. Adeno-LacZ treated NZM2328 showed enlarged glomeruli and increased cellularity without immune complex deposition. Adeno-LacZ treated R27 did not show serological and histological abnormalities. Adeno-IFNα induced anti-dsDNA and anti-kidney autoantibodies in NZM2328 and R27. These results suggest that end organ damage is host-dependent and less related to autoimmunity and may have significant implications in SLE pathogenesis.

Podocyte Activation of NLRP3 Inflammasomes Contributes to the Development of Proteinuria in Lupus Nephritis

Development of proteinuria in lupus nephritis (LN) is associated with podocyte dysfunction. The NLRP3 inflammasome has been implicated in the pathogenesis of LN. The purpose of this study was to investigate whether NLRP3 inflammasome activation is involved in the development of podocyte injury in LN.

Genetic Approach to Study Lupus Glomerulonephritis

Genetic and environmental factors contribute in the pathogenesis of systemic lupus erythematosus (SLE). Lupus nephritis, the most common and severe manifestation of SLE, involves inflammation in the kidney leading to loss of renal function. However, it is not clear what controls the progression of lupus nephritis; this is an important research question, considering its implications in clinical treatment of lupus nephritis. Finding genes that underlie the development and progression of lupus nephritis will shed light on this question. NZM2328 is a spontaneous mouse model for SLE. Most NZM2328 female mice develop autoantibodies (e.g., antinuclear antibody and anti-dsDNA antibody), glomerulonephritis (GN), and severe proteinuria between 5 and 12 months of age. In contrast, C57L/J mice fail to exhibit similar signs of autoimmune disease. We used classical genetics to map and identify SLE genes in offspring generated by backcrossing C57L/J to NZM2328. Quantitative trait loci (QTL) controlling acute (Agnz1 and Agnz2) and chronic (Cgnz1) GN features were uncovered by the analysis. To verify the Cgnz1 and Agnz1 on distal mouse chromosome 1, we produced the NZM23238.C57Lc1 (Lc1) congenic strain, which replaced NZM2328 Cgnz1 and Agnz1 alleles with those derived from C57L/J. The development of acute GN and chronic GN was markedly reduced in Lc1 mice, confirming the linkage findings. Further mapping by the generation of intrachromosomal recombinants of NZM2328.Lc1 support the thesis that acute GN and chronic GN are under separate genetic control.

Dependence of Glomerulonephritis Induction on Novel Intraglomerular Alternatively Activated Bone Marrow-Derived Macrophages and Mac-1 and PD-L1 in Lupus-Prone NZM2328 Mice.

Glomerular damage mediated by glomerulus-infiltrating myeloid-derived cells is a key pathogenic event in lupus nephritis (LN), but the process is poorly understood. Confocal microscopy of kidney sections and flow cytometry analysis of glomerular cells from magnetic bead–purified glomeruli have identified glomerulus-infiltrating leukocyte populations in NZM2328 (NZM) lupus-prone mice with spontaneous chronic glomerulonephritis (GN) and anti–glomerular basement membrane-induced nephritis. The occurrence of a major glomerulus-infiltrating CD11b+F4/80−I-A− macrophage population exhibiting the markers programmed death ligand-1 (PD-L1), Mac-2, and macrophage mannose receptor (CD206) and producing Klf4, Il10, Retnla, Tnf, and Il6 mRNA, which are known to be expressed by alternatively activated (M2b) macrophages, correlated with proteinuria status. In NZM mice with spontaneous LN, glomerular macrophage infiltration is predominant. CD11b+F4/80−I-A− intraglomerular macrophages and polymorphonuclear neutrophils (PMN) are important in inducing GN, as anti-CD11b and –ICAM-1 mAb inhibited both proteinuria and macrophage and PMN infiltration. The predominant and high expression of PD-L1 by CD11b+F4/80−I-A− glomerular macrophages in kidneys of mice with GN and the inhibition of proteinuria by anti–PD-L1 mAb supported the pathogenic role of these macrophages but not the PD-L1− PMN in GN development and in inducing podocyte damage. In NZM mice with spontaneous chronic GN and severe proteinuria, few glomerulus-infiltrating PMN were found, leaving macrophages and, to a less extent, dendritic cells as the major infiltrating leukocytes. Taken together, these data support the important pathogenic effect of CD11b+F4/80−I-A− M2b-like glomerulus-infiltrating macrophages in LN and reinforce macrophages as a promising target for GN treatment.

Nature of T cell epitopes in lupus antigens and HLA-DR determines autoantibody initiation and diversification

Objectives The generation of systemic lupus erythematosus (SLE)-related autoantibodies have been shown to be T cell dependent and antigen driven with HLA-DR restriction. In this study, the initiating antigen(s) and the mechanism of autoantibody diversification were investigated. Methods T cell epitopes (T-epitopes) of SmD1 (SmD) were mapped by T-T hybridomas generated from DR3+AE0 mice immunised with SmD and with SmD overlapping peptides. TCRs from the reactive hybridomas were sequenced. The core epitopes were determined. Bacterial mimics were identified by bioinformatics. Sera from DR3+AE0 mice immunised with SmD peptides and their mimics were analysed for their reactivity by ELISA and immunohistochemistry. Samples of blood donors were analysed for HLA-DR and autoantibody specificities. Results Multiple HLA-DR3 restricted T-epitopes within SmD were identified. Many T-T hybridomas reacted with more than one epitope. Some of them were cross-reactive with other snRNP peptides and with proteins in the Ro60/La/Ro52 complex. The reactive hybridomas used unique TCRs. Multiple T-epitope mimics were identified in commensal and environmental bacteria. Certain bacterial mimics shared both T and B cell epitopes with the related SmD peptide. Bacterial mimics induced autoantibodies to lupus-related antigens and to different tissues. HLA-DR3+ blood donors made significantly more SLE-related autoantibodies. Conclusions The unique antigenic structures of the lupus-related autoantigens provide the basis for being targeted and for T and B cell epitope spreading and autoantibody diversification with unique patterns. SLE-related autoantibodies are likely generated from responses to commensal and/or environmental microbes due to incomplete negative selection for autoreactive T cells. The production of SLE-related antibodies is inevitable in normal individuals. The findings in this investigation have significant implications in autoimmunity in general.

Mechanisms of Renal Damage in Systemic Lupus Erythematosus

Abstract Lupus nephritis (LN) is a major complication in systemic lupus erythematosus with considerable mortality and morbidities. The mechanisms of renal damage are complex. Since LN was considered a prototype of immune complex (IC)-mediated disease, major efforts have been focused on the nature of the IC with special emphasis on the nature of the targeted antigens (Ags). The prevailing view is that renal damage in LN is due to IC-mediated glomerulonephritis and anti-dsDNA antibodies–dsDNA IC is the most important system in the pathogenesis of LN. Recent studies suggest that this prevailing view should be reconsidered. Genetic studies provide evidence that anti-dsDNA antibodies and antinuclear antibodies such as antinucleosome antibodies are not required for the establishment of LN. Newer proteomic techniques demonstrated that anti-dsDNA antibodies are not the starters of LN and that antibodies to multiple podocyte antigens and planted antigens such as dsDNA and histones are involved. It is recognized that multiple cells and cytokines in addition to complement are involved in renal damage. The multitudes of mechanisms in renal damage in LN justify the multitarget approach in therapy. Recent genetic data support the thesis that end organ resistance to damage is under genetic control and that IC-mediated acute glomerulonephritis need not progress to chronic glomerulonephritis and end stage renal failure. These and other studies let us postulate that autoimmune response should be separated from end organ damage. The interaction of these two pathways leads to end organ damage. The model reminds us that the targeted organs in autoimmune diseases are active participants and their contribution to the pathology should be considered.

GG-13 Pathogenesis of lupus proliferative glomerulonephritis (LPGN): podocytes as targets and responders provide evidence for the importance of local factors controlling end organ damage

Background LPGN is the prototypic immune-complex (IC) mediated disease. The current dogma that IC deposition with complement (C’) activation inevitably leads to renal damage is neither sufficient nor comprehensive to account for the pathogenesis of LPGN. Our genetic studies support the thesis that acute GN (aGN) with IC deposition with C’ activation and cell infiltration and mesangial proliferation is a distinct phenotype from chronic GN (cGN) that is characterised by glomerulosclerosis, tubular dilatation and interstitial fibrosis with severe proteinuria and premature mortality. Furthermore, circulating ANA and anti-dsDNA Abs are not required for LPGN. Materials and methods Female mice of NZM2328 and its intra-chromosomal recombinant congenic line NZM2328.Lc1R27 (R27) were used. Anti-GBM induced GN was used as a model for IC mediated LPGN. Immunofluorescence was used to identify cell populations that made cytokines and complement components. Results R27 developed aGN and mild proteinuria without progression to cGN, end stage renal failure and early mortality. The kidneys of aged R27 had IC deposition and cellular infiltration, not distinguishable from that of aGN in NZM2328. Multiple approaches showed that the lack of progression from aGN to cGN in R27 was due to podocyte resistance to IC-mediated damage, a phenotype controlled by the allelic Cgnz1 gene. With a novel method to study intra-glomerular cytokine production, NZM podocytes were shown to be the major cell population that makes IL-1β in cGN, infiltrating CD11b+ macrophages make TNFα and the mesangial cells make IL-6. R27 mice do not show this compartmentalization of cytokine production. Preliminary data showed that the podocytes in Class III and IV lupus GN make IL-1β. Podocytes at the early and late cGN were shown to make C1q and C3. The expression of these complement components is less evident in R27 kidneys. C1q and C3 were present in some podocytes in biopsies of class III and IV lupus nephritis. Urinary podocytes making C1q were detected by us in of four patients with LPGN but not in normal individuals. Conclusions Our studies of lupus GN in both mouse and in man have provided significant information and insight regarding the role of podocytes as targets and as responders to IC mediated injuries. Our results suggest that the pathogenesis of LPGN should be revisited with focus on the local factors that may be of paramount importance. In addition to enhance our basic knowledge of podocyte biology, our results may provide novel targets for intervention and new urinary biomarkers to monitor therapeutic responses.