Novel mechanism for estrogen receptor alpha modulation of murine lupus.

Female sex is a risk factor for lupus. Sex hormones, sex chromosomes and hormone receptors are implicated in the pathogenic pathways in lupus. Estrogen receptor alpha (ERα) knockout (KO) mice are used for defining hormone receptor effects in lupus. Prior studies of ERα KO in lupus have conflicting results, likely due to sex hormone levels, different lupus strains and different ERα KO constructs. Our objective was to compare a complete KO of ERα vs. the original functional KO of ERα (expressing a short ERα) on disease expression and immune phenotype, while controlling sex hormone levels. We studied female lupus prone NZM2410 WT and ERα mutant mice. All mice (n = 44) were ovariectomized (OVX) for hormonal control. Groups of each genotype were estrogen (E2)-repleted after OVX. We found that OVXed NZM mice expressing the truncated ERα (ERα short) had significantly reduced nephritis and prolonged survival compared to both wildtype and the complete ERαKO (ERα null) mice, but surprisingly only if E2-repleted. ERα null mice were not protected regardless of E2 status. We observed significant differences in splenic B cells and dendritic cells and a decrease in cDC2 (CD11b+CD8-) dendritic cells, without a concomitant decrease in cDC1 (CD11b-CD8a+) cells comparing ERα short to ERα null or WT mice. Our data support a protective role for the ERα short protein. ERα short is similar to an endogenously expressed ERα variant (ERα46). Modulating its expression/activity represents a potential approach for treating female-predominant autoimmune diseases.

Complete knockout of estrogen receptor alpha is not directly protective in murine lupus.

Systemic lupus erythematosus (SLE) is a chronic and potentially severe autoimmune disease that disproportionately affects women. Despite a known role for hormonal factors impacting autoimmunity and disease pathogenesis, the specific mechanisms of action remain poorly understood. Our laboratory previously backcrossed "estrogen receptor alpha knockout (ERαKO)" mice onto the NZM2410 lupus prone background to generate NZM/ERαKO mice. This original ERαKO mouse, developed in the mid-1990s and utilized in hundreds of published studies, is not in fact ERα null. They express an N-terminally truncated ERα, and are considered a functional KO. They have physiologic deficiencies including infertility due to disruption of a critical activation domain (AF-1) at the N terminus of ERα, required for most classic estrogen (E2) actions. We demonstrated that female NZM/ERαKO mice had significantly less renal disease and significantly prolonged survival compared to WT littermates despite similar serum levels of autoantibodies and glomerular immune complex deposition. Herein, we present results of experiments using a lupus prone true ERα-/- mice (deletional KO mice on the NZM2410 background), surprisingly finding that these animals were not protected if they were ovariectomized, suggesting that another hormonal component confers protection, possibly testosterone, rather than the absence of the full-length ERα.

The podocyte: glomerular sentinel at the crossroads of innate and adaptive immunity.

Focal segmental glomerulosclerosis (FSGS) is a common glomerular disorder that manifests clinically with the nephrotic syndrome and has a propensity to recur following kidney transplantation. The pathophysiology and therapies available to treat FSGS currently remain elusive. Since the podocyte appears to be the target of apparent circulating factor(s) that lead to recurrence of proteinuria following kidney transplantation, this article is focused on the podocyte. In the context of kidney transplantation, the performance of pre- and post-reperfusion biopsies, and the establishment of in vitro podocyte liquid biopsies/assays allow for the development of clinically relevant studies of podocyte biology. This has given insight into new pathways, involving novel targets in innate and adaptive immunity, such as SMPDL3b, cGAS-STING, and B7-1. Elegant experimental studies suggest that the successful clinical use of rituximab and abatacept, two immunomodulating agents, in our case series, may be due to direct effects on the podocyte, in addition to, or perhaps distinct from their immunosuppressive functions. Thus, tissue biomarker-directed therapy may provide a rational approach to validate the mechanism of disease and allow for the development of new therapeutics for FSGS. This report highlights recent progress in the field and emphasizes the importance of kidney transplantation and recurrent FSGS (rFSGS) as a platform for the study of primary FSGS.

TLR7 Agonism Accelerates Disease and Causes a Fatal Myeloproliferative Disorder in NZM 2410 Lupus Mice.

Murine models of lupus, both spontaneous and inducible, are valuable instruments to study SLE pathogenesis. Accelerants such as Type I IFN are often used to trigger earlier disease onset. We used a topical TLR7 agonist, previously reported to induce lupus-like disease in WT mice within weeks, to validate this data in C57BL/6j mice, and to test TLR7 agonism as an accelerant in lupus-prone NZM2410 mice. We found that TLR7-stimulated B6 and NZM2410 mice had significantly reduced survival and exhibited profound splenomegaly with significantly reduced B cells (4 vs. 40%), and T cells (8 vs. 31%). Spleen pathology and IHC revealed massive expansion of F4/80+ cells in TLR7-treated mice consistent with histiocytosis. While resiqimod treatment caused mild autoimmunity in B6 mice and accelerated autoimmunity in NZM2410 mice, it did not cause significant nephritis or proteinuria in either strain (renal function intact at death). Given the macrophage expansion, cytopenias, and disruption of normal splenic lymphoid follicle architecture, histiocytic sarcoma is favored as the cause of death. An alternative etiology is a macrophage activation syndrome (MAS)-like syndrome, since the mice also had a transaminitis and histologic hemophagocytosis in the setting of their rapid mortality. For investigators who are focused on murine models of lupus nephritis, this model is not ideal when utilizing B6 mice, however topical resiqimod may prove useful to accelerate autoimmunity and nephritis in NZM2410 mice, or potentially to investigate secondary complications of lupus such as histiocytic diseases or macrophage activation like syndromes.

Impact of estrogen receptor deficiency on disease expression in the NZM2410 lupus prone mouse.

Systemic lupus erythematosus (SLE) occurs nine times more often in females than males. The purpose of this study was to determine the impact of estrogen receptor (ER) null genotypes on disease in lupus prone NZM2410 (NZM) and MRL/lpr mice, as a method to define the role of estrogen receptor signaling in lupus. ER alpha deficient NZM females, but not males, had significantly prolonged survival, reduced proteinuria, renal pathology scores and serum urea nitrogen levels compared to wildtype mice, despite higher serum anti-dsDNA levels. ER alpha deficient MRL/lpr female, but not male, mice also had significantly less proteinuria and renal pathology scores with no effect on autoantibody levels. Deficiency of ER beta had no effect on disease in either strain or sex. Taken together, these data demonstrate a key role for ER alpha, but not ER beta, in the development of lupus like disease, but not autoimmunity, in female NZM and MRL/lpr mice.

Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse.

Studies in human systemic lupus erythematosus (SLE) suggest a possible role for histone deacetylases (HDACs) in skewed gene expression and disease pathogenesis. We used the MRL-lpr/lpr murine model of lupus to demonstrate that HDACs play a key role in the heightened levels of both Th1 and Th2 cytokine expression that contribute to disease. The availability of specific HDAC inhibitors (HDIs) such as trichostatin A (TSA) and suberonylanilide hydroxamic acid (SAHA) permits the study of the role of HDACs in gene regulation. Our results indicate that HDIs downregulate IL-12, IFN-gamma, IL-6, and IL-10 mRNA and protein levels in MRL-lpr/lpr splenocytes. This effect on gene transcription is associated with an increased accumulation of acetylated histones H3 and H4 in total cellular chromatin. To elucidate the in vivo effects of TSA on lupuslike disease, we treated MRL-lpr/lpr mice with TSA (0.5 mg/kg/d) for 5 weeks. Compared with vehicle-treated control mice, TSA-treated mice exhibited a significant reduction in proteinuria, glomerulonephritis, and spleen weight. Taken together, these findings suggest that increased expression of HDACs leading to an altered state of histone acetylation may be of pathologic significance in MRL-lpr/lpr mice. In addition, TSA or other HDIs may have therapeutic benefit in the treatment of SLE.

Liver biopsy interpretation for causes of late liver allograft dysfunction.

Evaluation of needle biopsies and extensive clinicopathological correlation play an important role in the determination of liver allograft dysfunction occurring more than 1 year after transplantation. Interpretation of these biopsies can be quite difficult because of the high incidence of recurrent diseases that show histopathological, clinical, and serological features that overlap with each other and with rejection. Also, more than one insult can contribute to allograft injury. In an attempt to enable centers to compare and pool results, improve therapy, and better understand pathophysiological disease mechanisms, the Banff Working Group on Liver Allograft Pathology herein proposes a set of consensus criteria for the most common and problematic causes of late liver allograft dysfunction, including late-onset acute and chronic rejection, recurrent and new-onset viral and autoimmune hepatitis, biliary strictures, and recurrent primary biliary cirrhosis and primary sclerosing cholangitis. A discussion of differential diagnosis is also presented.

Effects of complement factor D deficiency on the renal disease of MRL/lpr mice.

BACKGROUND: The alternative complement pathway (AP) is activated in individuals with lupus nephritis and in murine models of systemic lupus erythematosus, including MRL/lpr mice. A previous study from our laboratory evaluated the development of renal disease in MRL/lpr mice genetically deficient in factor B (Bf-/-), a protein necessary for AP activation. MRL/lpr Bf-/- mice developed less renal disease and had improved survival; however, these mice were also a different major histocompatibility complex (MHC) haplotype (H-2b) than their wild-type littermates (H-2k) due to the gene for Bf being located in the MHC gene complex. We undertook the current study to determine if the decreased renal disease in MRL/lpr Bf-/- mice was due to the lack of AP activation or the H-2b haplotype by studying the effects of factor D (Df) deficiency, a critical protein for AP activation, on disease development in MRL/lpr mice. METHODS: Df-deficient mice were backcrossed with MRL/lpr mice for four to nine generations. MRL/lpr H-2k Df-/-, Df+/-, and Df+/+ littermates were evaluated for disease development. Lack of AP activation in MRL/lpr Df-/- mice was determined by the zymosan assay. Serum creatinine levels were measured using a creatinine kit. Proteinuria and autoantibody levels were determined by enzyme-linked immunosorbent assay (ELISA). Sections from one kidney were stained with fluorescein isothiocyanate (FITC) alpha-murine C3 or alpha-murine IgG to detect C3 and IgG deposition. The remaining kidney was cut in half with one half fixed, sectioned, and stained with hematoxylin and eosin and periodic acid-Schiff (PAS) to evaluate pathology and another half fixed in glutaraldehyde and examined via electron microscopy. RESULTS: MRL/lpr Df-/- mice had similar glomerular IgG deposition, proteinuria and autoantibody levels, as Df+/+ and Df+/- littermates. However, glomerular C3 deposition, serum creatinine levels, and pathologic renal disease were significantly reduced in Df-/- mice. Despite the lack of renal disease in Df-/- mice, life span was not impacted by factor D deficiency. CONCLUSION: The absence of Df and AP activation is protective against the development of proliferative renal disease in MRL/lpr mice suggesting the similar effect of Bf deficiency in MRL/lpr mice was also due to the lack of AP activation.

Effect of genetic deficiency of terminal deoxynucleotidyl transferase on autoantibody production and renal disease in MRL/lpr mice.

Terminal deoxynucleotidyl transferase (TdT) places non-template-coded nucleotides (N additions) in the VH CDR3 of T cell receptors and immunoglobulins. Amino acids coded for by N additions are important in autoantibody binding of dsDNA in lupus. We hypothesized that a genetic lack of TdT would modulate disease in lupus-prone mice. To test this hypothesis, we derived TdT-deficient MRL/lpr mice. Serum levels of anti-dsDNA antibodies and anti-dsDNA producing splenocytes were significantly lower in the TdT(-) versus TdT(+) littermates. Albuminuria, glomerular IgG deposition, and pathologic renal disease were significantly reduced in the TdT(-) mice. Sequence analysis of anti-dsDNA hybridomas derived from TdT(-) mice revealed a lack of N additions, short VH CDR3 segments, yet the presence of VH CDR3 arginines. Thus, the genetic absence of TdT reduces autoantibody production and clinical disease in MRL/lpr mice, confirming the importance of N additions in the autoimmune response in these mice.

Modulation of renal disease in MRL/lpr mice by pharmacologic inhibition of inducible nitric oxide synthase.

BACKGROUND: MRL-MPJFaslpr (MRL/lpr) mice spontaneously develop lupus-like disease characterized by immune complex glomerulonephritis and overproduction of nitric oxide (NO). Blocking NO production pharmacologically by a non-specific nitric oxide synthase (NOS) inhibitor ameliorated renal disease in MRL/lpr mice while genetically deficient inducible NOS (iNOS) mice developed proliferative glomerulonephritis similar to wild-type controls. METHODS: To clarify the role of iNOS in the pathogenesis of nephritis in MRL/lpr mice, we treated mice with two different NOS inhibitors. Either NG-monomethyl-l-arginine (L-NMMA), a nonspecific NOS inhibitor, or l-N6-(1-iminoethyl)lysine (L-NIL), an iNOS specific inhibitor, was administered in the drinking water from 10 through 22 weeks of age with disease progression monitored over time. Control mice received water alone. RESULTS: Both L-NMMA and L-NIL blocked NO production effectively in MRL/lpr mice. As expected, neither L-NNMA nor L-NIL had an effect on antibody production, immune complex deposition or complement activation. Although both NOS inhibitors decreased protein excretion, L-NMMA was more effective than L-NIL. Pathologic renal disease was significantly decreased at 19 weeks in both treatment groups. At 22 weeks the L-NIL treated mice, but not the L-NMMA mice, had significantly reduced renal disease scores compared to controls. CONCLUSION: These results indicate that specific inhibition of iNOS blocks the development of pathologic renal disease in MRL/lpr mice.