Distinct genome-wide DNA methylation and gene expression signatures in classical monocytes from African American patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a multisystem autoimmune disorder that has an unclear etiology and disproportionately affects women and African Americans. Despite this, African Americans are dramatically underrepresented in SSc research. Additionally, monocytes show heightened activation in SSc and in African Americans relative to European Americans. In this study, we sought to investigate DNA methylation and gene expression patterns in classical monocytes in a health disparity population. METHODS: Classical monocytes (CD14+ + CD16-) were FACS-isolated from 34 self-reported African American women. Samples from 12 SSc patients and 12 healthy controls were hybridized on MethylationEPIC BeadChip array, while RNA-seq was performed on 16 SSc patients and 18 healthy controls. Analyses were computed to identify differentially methylated CpGs (DMCs), differentially expressed genes (DEGs), and CpGs associated with changes in gene expression (eQTM analysis). RESULTS: We observed modest DNA methylation and gene expression differences between cases and controls. The genes harboring the top DMCs, the top DEGs, as well as the top eQTM loci were enriched for metabolic processes. Genes involved in immune processes and pathways showed a weak upregulation in the transcriptomic analysis. While many genes were newly identified, several other have been previously reported as differentially methylated or expressed in different blood cells from patients with SSc, supporting for their potential dysregulation in SSc. CONCLUSIONS: While contrasting with results found in other blood cell types in largely European-descent groups, the results of this study support that variation in DNA methylation and gene expression exists among different cell types and individuals of different genetic, clinical, social, and environmental backgrounds. This finding supports the importance of including diverse, well-characterized patients to understand the different roles of DNA methylation and gene expression variability in the dysregulation of classical monocytes in diverse populations, which might help explaining the health disparities.

Conditional knockout of oestrogen receptor alpha in CD11c+ cells impacts female survival and inflammatory cytokine profile in murine lupus.

Oestrogen and oestrogen receptor alpha (ERα) have been implicated in systemic lupus erythematosus pathogenesis. ERα signalling influences dendritic cell (DC) development and function, as well as inflammation and downstream immune responses. We previously reported that ERα modulates multiple Toll-like receptor-stimulated pathways in both conventional and plasmacytoid DCs in lupus-prone mice. For example, CD11chi MHCII+ cell numbers are reduced in mice with global ERα deficiency or when expressing a short variant of ERα. Herein, RNA-seq analysis of CD11chi cells from bone marrow of NZM2410 mice expressing WT ERα versus ERα short versus ERα null revealed differentially expressed complement genes, interferon-related genes and cytokine signalling (e.g., IL-17 and Th17 pathways). To better understand the role of ERα in CD11c+ cells, lupus prone NZM2410 mice with selective deletion of the Esr1 gene in CD11c+ cells were generated. Phenotype and survival of these mice were similar with the exception of Cre positive (CrePos) female mice. CrePos females, but not males, all died unexpectedly prior to 35 weeks. DC subsets were not significantly different between groups. Since ERα is necessary for robust development of DCs, this result suggests that DC fate was determined prior to CD11c expression and subsequent ERα deletion (i.e., proximally in DC ontogeny). Overall, findings point to a clear functional role for ERα in regulating cytokine signalling and inflammation, suggesting that further study into ERα-mediated regulatory mechanisms in DCs and other immune cell types is warranted.

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.

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.

Plasmacytoid dendritic cell distribution and maturation are altered in lupus prone mice prior to the onset of clinical disease.

Plasmacytoid dendritic cells (pDCs) and their production of type I interferons (IFN) are key pathogenic mediators of systemic lupus erythematosus (SLE). Despite the key role of pDCs in SLE, the mechanism by which pDCs promote disease is not well understood. The first objective for this study was to assess the number and maturation state of pDCs in pre-disease NZM2410 lupus prone mice compared to control mice. Second, we sought to identify mechanisms responsible for the alteration in pDCs in NZM mice prior to onset of clinical disease. We compared the number and percent of pDCs in the spleens and bone marrow (BM) of pre-disease NZM24010 (NZM) mice to C57BL/6 (B6) control mice. In the spleens of pre-disease NZM mice, pDC percent and number were increased. This increase occurs in parallel with a decrease in BM pDC number and percent in the NZM mice. The decrease in BM pDC number suggests the increase in spleen pDCs is a result of altered pDC distribution and not increased production of pDCs in the BM. To determine if pDC developmental potential is altered in lupus prone mice, we cultured BM from NZM and B6 mice in vitro. We found a reduced percentage/number of pDCs developing from the BM of NZM mice compared to B6 mice, which further supports that the increase in pDC number is a result of altered pDC distribution rather than increased pDC production. To better characterize the pDC population, we compared the percentage of mature pDCs in the spleens and BM of NZM mice to controls. In the NZM mice, there is a dramatic reduction in the number of mature pDCs in the BM of NZM mice, suggesting that mature pDCs exit the BM at a higher rate/earlier maturation time compared to healthy mice. We conclude that pDCs contribution to disease pathogenesis in NZM mice may include the alteration of pDC distribution to increase the number of pDCs in the spleen prior to disease onset.

Early Ovariectomy Results in Reduced Numbers of CD11c+/CD11b+ Spleen Cells and Impacts Disease Expression in Murine Lupus.

Ninety percent of those diagnosed with systemic lupus erythematosus are female, with peak incidence between the ages of 15 and 45, when women are most hormonally active. Despite significant research effort, the mechanisms underlying this sex bias remain unclear. We previously showed that a functional knockout of estrogen receptor alpha (ERα) resulted in significantly reduced renal disease and increased survival in murine lupus. Dendritic cell (DC) development, which requires both estrogen and ERα, is impacted, as is activation status and cytokine production. Since both estrogen and testosterone levels have immunomodulating effects, we presently studied the phenotype of NZM2410 lupus-prone mice following post- and prepubertal ovariectomy (OVX) ± estradiol (E2) replacement to determine the impact of hormonal status on disease expression and DC development in these mice. We observed a trend toward survival benefit in addition to decreased proteinuria and improved renal histology in the early OVX, but not late OVX- or E2-repleted WT mice. Interestingly, there was also a significant difference in splenic DC subsets by flow cytometry. Spleens from NZM mice OVX'd early had a significant decrease in proinflammatory CD11c+CD11b+ DCs (vs. unmanipulated WTs, late OVX- and E2-repleted mice). These early OVX'd animals also had a significant increase in tolerogenic CD11c+CD8a+ DCs vs. WT. These data join a growing body of evidence that supports a role for hormone modulation of DCs that likely impacts the penetrance and severity of autoimmune diseases, such as lupus.

Estrogen Receptor α Deficiency Modulates TLR Ligand-Mediated PDC-TREM Expression in Plasmacytoid Dendritic Cells in Lupus-Prone Mice.

Female lupus-prone NZM2410 estrogen receptor α (ERα)-deficient mice are protected from renal disease and have prolonged survival compared with wild-type littermates; however, the mechanism of protection is unknown. Plasmacytoid dendritic cells (pDCs) and type I IFN drive lupus pathogenesis. Estrogen acting via ERα enhances both pDC development and IFN production. The objectives for this study were to determine if ERα modulates pDC function and IFN activity in predisease NZM2410 mice as a possible protective mechanism of ERα deficiency in lupus-prone mice. We measured the effect of ERα deficiency on spleen pDC frequency, number, maturation, and activation state. ERα deficiency reduced type I IFN activity and the frequency of MHC class II(+) pDCs in the spleen without altering overall pDC frequency, number, or maturation state. Additionally, ERα-deficient NZM2410 mice had a significantly decreased frequency of pDCs expressing PDC-TREM, a modulator of TLR-mediated IFN production. After in vitro TLR9 stimulation, ERα deficiency significantly reduced the expression of PDC-TREM on pDCs from both NZM2410 and C57BL/6 mice. Thus, we have identified a significant effect of ERα deficiency on pDCs in predisease NZM2410 mice, which may represent a mechanism by which ERα deficiency protects NZM2410 mice from lupuslike disease.

Biaryls as potent, tunable dual neurokinin 1 receptor antagonists and serotonin transporter inhibitors.

Depression is a serious illness that affects millions of patients. Current treatments are associated with a number of undesirable side effects. Neurokinin 1 receptor (NK1R) antagonists have recently been shown to potentiate the antidepressant effects of serotonin-selective reuptake inhibitors (SSRIs) in a number of animal models. Herein we describe the optimization of a biaryl chemotype to provide a series of potent dual NK1R antagonists/serotonin transporter (SERT) inhibitors. Through the choice of appropriate substituents, the SERT/NK1R ratio could be tuned to afford a range of target selectivity profiles. This effort culminated in the identification of an analog that demonstrated oral bioavailability, favorable brain uptake, and efficacy in the gerbil foot tap model. Ex vivo occupancy studies with compound 58 demonstrated the ability to maintain NK1 receptor saturation (>88% occupancy) while titrating the desired level of SERT occupancy (11-84%) via dose selection.

Estrogen receptor alpha deficiency protects against development of cognitive impairment in murine lupus.

BACKGROUND: One of the more profound features of systemic lupus erythematosus (SLE) is that females have a 9:1 prevalence of this disease over males. Up to 80% of SLE patients have cognitive defects or affective disorders. The mechanism of CNS injury responsible for cognitive impairment is unknown. We previously showed that ERα deficiency significantly reduced renal disease and increased survival in lupus-prone mice. We hypothesized that ERα deficiency would be similarly protective in the brain, and that ERα may play a role in modulating blood-brain barrier (BBB) integrity and/or neuroinflammation in lupus-prone mice. METHODS: MRL/lpr ERα+/+ and ERαKO mice (n = 46) were ovariectomized, received 17ß-estradiol pellets, and underwent radial arm water maze (WRAM) and novel object recognition (NOR) testing starting at eight weeks of age. Mice were sacrificed and brains were hemisected and processed for either immunohistochemistry, or hippocampus and parietal cortex dissection for Western blotting. RESULTS: MRL/lpr ERαKO mice (n = 21) performed significantly better in WRAM testing than wild-type MRL/lpr mice (n = 25). There was a significant reduction in reference memory errors (P <0.007), working memory errors (P <0.05), and start arm errors (P <0.02) in ERαKO mice. There were significant differences in NOR testing, particularly total exploration time, with ERα deficiency normalizing behavior. No significant differences were seen in markers of tight junction, astrogliosis, or microgliosis in the hippocampus or cortex by Western blot, however, there was a significant reduction in numbers of Iba1+ activated microglia in the hippocampus of ERαKO mice, as evidenced by immunohistochemietry (IHC). CONCLUSION: ERα deficiency provides significant protection against cognitive deficits in MRL/lpr mice as early as eight weeks of age. Additionally, the significant reduction in Iba1+ activated microglia in the MRL/lpr ERαKO mice was consistent with reduced inflammation, and may represent a biological mechanism for the cognitive improvement observed.

Optimal management of fatigue in patients with systemic lupus erythematosus: a systematic review.

Among the host of distressing pathophysiological and psychosocial symptoms, fatigue is the most prevalent complaint in patients with systemic lupus erythematosus (SLE). This review is to update the current findings on non-pharmacological, pharmacological, and modality strategies to manage fatigue in patients with SLE and to provide some recommendations on optimal management of fatigue based on the best available evidence. We performed a systematic literature search of the PubMed and Scopus databases to identify publications on fatigue management in patients with SLE. Based on the studies reported in the literature, we identified nine intervention strategies that have the potential to alleviate fatigue in patients with SLE. Of the nine strategies, aerobic exercise and belimumab seem to have the strongest evidence of treatment efficacy. N-acetylcysteine and ultraviolet-A1 phototherapy demonstrated low-to-moderate levels of evidence. Psychosocial interventions, dietary manipulation (low calorie or glycemic index diet) aiming for weight loss, vitamin D supplementation, and acupuncture all had weak evidence. Dehydroepiandrosterone is not recommended due to a lack of evidence for its efficacy. In addition to taking treatment efficacy and side effects into consideration, clinicians should consider factors such as cost of treatment, commitments, and burden to the patient when selecting fatigue management strategies for patients with SLE. Any comorbidities, such as psychological distress, chronic pain, sleep disturbance, obesity, or hypovitaminosis D, associated with fatigue should be addressed.

Estrogen Receptor Alpha Binding to ERE is Required for Full Tlr7- and Tlr9-Induced Inflammation.

We previously found that a maximum innate inflammatory response induced by stimulation of Toll-like receptors (TLRs) 3, 7 and 9 requires ERα, but does not require estrogen in multiple cell types from both control and lupus-prone mice. Given the estrogen-independence, we hypothesized that ERα mediates TLR signaling by tethering to, and enhancing, the activity of downstream transcription factors such as NFκB, rather than acting classically by binding EREs on target genes. To investigate the mechanism of ERα impact on TLR signaling, we utilized mice with a knock-in ERα mutant that is unable to bind ERE. After stimulation with TLR ligands, both ex vivo spleen cells and bone marrow-derived dendritic cells (BM-DCs) isolated from mutant ERα ("KIKO") mice produced significantly less IL-6 compared with cells from wild-type (WT) littermates. These results suggest that ERα modulation of TLR signaling does indeed require ERE binding for its effect on the innate immune response.

IgG4-Related Disease Presenting as Recurrent Mastoiditis With Central Nervous System Involvement.

We report a case of a 43-year-old female who presented with right ear fullness and otorrhea. She was initially diagnosed with mastoiditis that was not responsive to multiple courses of antibiotics and steroids. She was then diagnosed with refractory inflammatory pseudotumor, and subsequent treatments included several mastoidectomies, further steroids, and radiation therapy. The patient went on to develop mastoiditis on the contralateral side as well as central nervous system involvement with headaches and right-sided facial paresthesias. Reexamination of the mastoid tissue revealed a significantly increased number of IgG4-positive cells, suggesting a diagnosis of IgG4-related disease. The patient improved clinically and radiographically with rituximab and was able to taper off azathioprine and prednisone. IgG4-related disease should be considered in patients with otologic symptoms and be on the differential diagnosis in patients with inflammatory pseudotumor. Staining for IgG and IgG4 is essential to ensure a prompt diagnosis and treatment.

Estrogen receptor alpha modulates Toll-like receptor signaling in murine lupus.

Systemic lupus erythematosus (SLE) is a disease that disproportionately affects females. Despite significant research effort, the mechanisms underlying the female predominance in this disease are largely unknown. Previously, we showed that estrogen receptor alpha knockout (ERαKO) lupus prone female mice had significantly less pathologic renal disease and proteinuria, and significantly prolonged survival. Since autoantibody levels and number and percentage of B/T cells were not significantly impacted by ERα genotype, we hypothesized that the primary benefit of ERα deficiency in lupus nephritis was via modulation of the innate immune response. Using BMDCs and spleen cells/B cells from female wild-type or ERαKO mice, we found that ERαKO-derived cells have a significantly reduced inflammatory response after stimulation with TLR agonists. Our results indicate that the inflammatory response to TLR ligands is significantly impacted by the presence of ERα despite the absence of estradiol, and may partially explain the protective effect of ERα deficiency in lupus-prone animals.

Conformationally restricted homotryptamines. 2. Indole cyclopropylmethylamines as selective serotonin reuptake inhibitors.

A series of indole cyclopropylmethylamines were found to be potent serotonin reuptake inhibitors. Nitrile substituents at the 5 and 7 positions of the indole ring gave high affinity for hSERT, and the preferred cyclopropane stereochemistry was determined to be (1S,2S)-trans. The cis-cyclopropanes had 20- to 30-fold less affinity than the trans, and the preferred cis stereochemistry was (1R,2S)-cis. Substitution of the indole N-1 position with methyl or ethyl groups gave a 10- to 30-fold decrease in affinity for hSERT, suggesting either a hydrogen-bonding interaction or limited steric tolerance in the region of the indole nitrogen. Compound (+)-12a demonstrated potent hSERT binding (Ki = 0.18 nM) in vitro and was more than 1000-fold less potent at hDAT, hNET, 5-HT1A, and 5-HT6. In vivo, (+)-12a produced robust, dose-dependent increases in extracellular serotonin in rat frontal cortex typical of a selective serotonin reuptake inhibitor. The maximal response produced by (+)-12a was similar to that of fluoxetine but at an approximately 10-fold lower dose.

FoxO1a can alter cell cycle progression by regulating the nuclear localization of p27kip in granulosa cells.

Forkhead transcription factors of the FOXO family are important downstream targets of the phosphatidylinositol 3-kinase pathway, which has been shown to play a critical role in cell proliferation and cell survival. Activation of FOXOs can block cellular proliferation and drive cells into a quiescent state. In certain cell types, this cell cycle arrest is dependent on the transcriptional induction of the cell-cycle inhibitor p27kip. In granulosa cells, which go through an exponential growth phase during development of the ovarian follicle, we find that FoxO1a is a key regulator of the G1/S transition in these cells. Overexpression of a dominant-negative version of FoxO1a (Foxo1a-Delta256; a C-terminal truncation mutant that possesses a functional DNA-binding domain, but lacks a transactivation domain) causes a dramatic increase in S-phase cells (>8-fold increase by both DNA content and bromodeoxyuridine incorporation assays). Surprisingly, this is not dependent on transactivation of the p27kip gene. We provide evidence that when FoxO1a activity is impeded, p27kip protein is largely localized to the cytosol, suggesting that FoxO1a blocks cell cycle entry by altering the compartmentalization of p27kip within the cell, increasing its concentration in the nucleus. These studies demonstrate for the first time that FoxO1a can regulate p27kip nuclear localization.

Follicle-stimulating hormone promotes nuclear exclusion of the forkhead transcription factor FoxO1a via phosphatidylinositol 3-kinase in porcine granulosa cells.

The forkhead family of transcription factors is conserved in evolution and known to play critical roles in the regulation of cellular differentiation and proliferation in many systems. The current studies demonstrate for the first time that forkhead homolog in rhabdomyosarcoma (FKHR) (FoxO1a) is expressed in porcine granulosa cells, and FSH stimulates FKHR phosphorylation and regulates its subcellular localization in this system. RT-PCR and Western blot studies demonstrated that FKHR is expressed and showed no change in FKHR message or protein levels in response to FSH (0-6 h). However, [32p]-orthophosphate labeling of cultured granulosa cells revealed robust phosphorylation after FSH treatment for 30 min. In addition, FSH caused nuclear exclusion of FKHR in these cells, apparently through the phosphatidylinositol 3-kinase signal transduction pathway. The cytosolic accumulation of FKHR protein that was observed in FSH-treated cells both by Western blot and immunohistochemistry was blocked when the cells were preincubated with the phosphatidylinositol 3-kinase inhibitor LY294002. Our data also demonstrate that Akt/protein kinase B, an established kinase for FKHR, is phosphorylated in response to FSH treatment. Interestingly, although FKHR was phosphorylated by 30 min after FSH treatment, the time course for Akt phosphorylation was relatively delayed and sustained. Although these studies do not preclude Akt involvement in FSH-stimulated FKHR phosphorylation, they do suggest that other kinases may contribute to rapid signaling to FKHR. Because FKHR has been shown to activate genes involved in apoptosis and growth inhibition, FSH may promote growth and survival by initiating the phosphorylation of FKHR, causing its nuclear exclusion, and reducing its effect as a cell cycle arrest or death-promoting transcription factor.