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OBJECTIVE: About 3% of patients with lupus develop severe diffuse alveolar hemorrhage (DAH) with pulmonary vasculitis. C57BL/6 (B6) mice with pristane-induced lupus also develop DAH, but BALB/c mice are resistant. DAH is independent of Toll-like receptor signaling and other inflammatory pathways. This study examined the role of the MEK1/2 pathway (MEK1/2-ERK1/2, JNK, p38). METHODS: B6 and BALB/c mice were treated with pristane with or without inhibitors of MEK1/2 (trametinib/GSK1120212 [GSK]), ERK1/2 (SCH772984 [SCH]), JNK, or p38. Effects on lung hemorrhage and hemostasis were determined. RESULTS: GSK and SCH abolished DAH, whereas JNK and p38 inhibitors were ineffective. Apoptotic cells were present in lung samples from pristane-treated mice but not in mice receiving pristane and GSK, and endothelial dysfunction was normalized. Expression of the ERK1/2-regulated transcription factor early growth response 1 increased in pristane-treated B6, but not BALB/c, mice and was normalized by GSK. Pristane also increased expression of the anticoagulant genes Tfpi and Thbd in B6 mice. The ratio of Tfpi to tissue factor (F3) to Tfpi increased in B6 (but not BALB/c) mice and was normalized by GSK. Circulating thrombomodulin protein levels increased in B6 mice and returned to normal after GSK treatment. Consistent with augmented endothelial anticoagulant activity, pristane treatment increased tail bleeding in B6 mice. CONCLUSION: Pristane treatment promotes lung endothelial injury and DAH in B6 mice by activating the MEK1/2-ERK1/2 pathway and impairing hemostasis. The hereditary factors determining susceptibility to lung injury and bleeding in pristane-induced lupus are relevant to the pathophysiology of life-threatening DAH in systemic lupus erythematosus and may help to optimize therapy.
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Objective: About 3% of lupus patients develop severe diffuse alveolar hemorrhage (DAH) with pulmonary vasculitis. B6 mice with pristane-induced lupus also develop DAH, but BALB/c mice are resistant. DAH is independent of TLR signaling and other inflammatory pathways. This study examined the role of the mitogen-activated protein kinase pathway (MEK1/2-ERK1/2, JNK, p38). Methods: B6 and BALB/c mice were treated with pristane ± inhibitors of MEK1/2 (trametinib/GSK1120212, "GSK"), ERK1/2 (SCH772984, "SCH"), JNK, or p38. Effects on lung hemorrhage and hemostasis were determined. Results: GSK and SCH abolished DAH, whereas JNK and p38 inhibitors were ineffective. Apoptotic cells were present in lung from pristane-treated mice, but not mice receiving pristane+GSK and endothelial dysfunction was normalized. Expression of the ERK1/2-regulated transcription factor Egr1 increased in pristane-treated B6, but not BALB/c, mice and was normalized by GSK. Pristane also increased expression of the anticoagulant genes Tfpi (tissue factor pathway inhibitor) and Thbd (thrombomodulin) in B6 mice. The ratio of tissue factor ( F3 ) to Tfpi increased in B6 (but not BALB/c) mice and was normalized by GSK. Circulating Thbd protein increased in B6 mice and returned to normal after GSK treatment. Consistent with augmented endothelial anticoagulant activity, pristane treatment increased tail bleeding in B6 mice. Conclusion: Pristane treatment promotes lung endothelial injury and DAH in B6 mice by activating the MEK1/2-ERK1/2 pathway and impairing hemostasis. The hereditary factors determining susceptibility to lung injury and bleeding in pristane-induced lupus are relevant to the pathophysiology of life-threatening DAH in SLE and may help to optimize therapy.
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Oxidants participate in lymphocyte activation and function. We previously demonstrated that eliminating the activity of NADPH oxidase 2 (NOX2) significantly impaired the effectiveness of autoreactive CD8+ CTLs. However, the molecular mechanisms impacting CD8+ T cell function remain unknown. In the present study, we examined the role of NOX2 in both NOD mouse and human CD8+ T cell function. Genetic ablation or chemical inhibition of NOX2 in CD8+ T cells significantly suppressed activation-induced expression of the transcription factor T-bet, the master transcription factor of the Tc1 cell lineage, and T-bet target effector genes such as IFN-γ and granzyme B. Inhibition of NOX2 in both human and mouse CD8+ T cells prevented target cell lysis. We identified that superoxide generated by NOX2 must be converted into hydrogen peroxide to transduce the redox signal in CD8+ T cells. Furthermore, we show that NOX2-generated oxidants deactivate the tumor suppressor complex leading to activation of RheB and subsequently mTOR complex 1. These results indicate that NOX2 plays a nonredundant role in TCR-mediated CD8+ T cell effector function.
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Linfocitos T CD8-positivos , NADPH Oxidasa 2 , Especies Reactivas de Oxígeno , Animales , Humanos , Ratones , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , Granzimas/metabolismo , Peróxido de Hidrógeno/metabolismo , Inflamación/inmunología , Interferón gamma/metabolismo , Activación de Linfocitos , Ratones Endogámicos NOD , NADPH Oxidasa 2/antagonistas & inhibidores , NADPH Oxidasa 2/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Proteínas de Dominio T Box/metabolismo , Masculino , Femenino , Adulto JovenRESUMEN
Pristane causes chronic peritoneal inflammation resulting in lupus, which in C57BL/6 mice is complicated by lung microvascular injury and diffuse alveolar hemorrhage (DAH). Mineral oil (MO) also causes inflammation, but not lupus or DAH. Since monocyte depletion prevents DAH, we examined the role of monocytes in the disease. Impaired bone marrow (BM) monocyte egress in Ccr2-/- mice abolished DAH, confirming the importance of monocyte recruitment to the lung. Circulating Ly6Chi monocytes from pristane-treated mice exhibited increased annexin-V staining in comparison with MO-treated controls without evidence of apoptosis, suggesting that pristane alters the distribution of phosphatidylserine in the plasma membrane before or shortly after monocyte egress from the BM. Plasma membrane asymmetry also was impaired in Nr4a1-regulated Ly6Clo/- 'patrolling' monocytes, which are derived from Ly6Chi precursors. Patrolling Ly6Clo/- monocytes normally promote endothelial repair, but their phenotype was altered in pristane-treated mice. In contrast to MO-treated controls, Nr4a1-regulated Ly6Clo/- monocytes from pristane-treated mice were CD138+, expressed more TremL4, a protein that amplifies TLR7 signaling, and exuberantly produced TNFα in response to TLR7 stimulation. TremL4 expression on these novel CD138+ monocytes was regulated by Nr4a1. Thus, monocyte CD138, high TremL4 expression, and annexin-V staining may define an activated/inflammatory subtype of patrolling monocytes associated with DAH susceptibility. By altering monocyte development, pristane exposure may generate activated Ly6Chi and Ly6Clo/- monocytes, contributing to lung microvascular endothelial injury and DAH susceptibility.
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Enfermedades Pulmonares , Monocitos , Ratones , Animales , Monocitos/metabolismo , Ratones Endogámicos C57BL , Factor de Necrosis Tumoral alfa/metabolismo , Aceite Mineral/metabolismo , Fosfatidilserinas/metabolismo , Receptor Toll-Like 7/metabolismo , Enfermedades Pulmonares/metabolismo , Hemorragia/inducido químicamente , Inflamación/metabolismo , Anexinas/metabolismoRESUMEN
Human COPA mutations affecting retrograde Golgi-to-endoplasmic reticulum (ER) protein transport cause diffuse alveolar hemorrhage (DAH) and ER stress ("COPA syndrome"). Patients with SLE also can develop DAH. C57BL/6 (B6) mice with pristane-induced lupus develop monocyte-dependent DAH indistinguishable from human DAH, whereas BALB/c mice are resistant. We examined Copa and ER stress in pristane-induced lupus. Copa expression, ER stress, vascular injury, and apoptosis were assessed in mice and COPA was quantified in blood from patients with SLE. Copa mRNA and protein expression were impaired in B6 mice with pristane-induced DAH, but not in pristane-treated BALB/c mice. An ER stress response (increased Hsp5a/BiP, Ddit3/CHOP, Eif2a, and spliced Xbp1) was seen in lungs from pristane-treated B6, but not BALB/c, mice. Resistance of BALB/c mice to DAH was overcome by treating them with low-dose thapsigargin plus pristane. CB6F1 mice did not develop DAH or ER stress, suggesting that susceptibility was recessive. Increased pulmonary expression of von Willebrand factor (Vwf), a marker of endothelial injury, and the chemokine Ccl2 in DAH suggested that pristane promotes lung microvascular injury and monocyte recruitment. Consistent with that possibility, lung endothelial cells and infiltrating bone marrow-derived cells from pristane-treated B6 mice expressed BiP and showed evidence of apoptosis (annexin-V and activated caspase-3 staining). COPA expression also was low in patients with SLE with lung involvement. Pristane-induced DAH may be initiated by endothelial injury, resulting in ER stress, apoptosis of lung endothelial cells, and recruitment of myeloid cells that propagate lung injury. The pathogenesis of DAH in SLE and COPA syndrome may overlap.
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Enfermedades Pulmonares , Lesión Pulmonar , Lupus Eritematoso Sistémico , Vasculitis , Humanos , Ratones , Animales , Alveolos Pulmonares/patología , Lesión Pulmonar/patología , Células Endoteliales/patología , Ratones Endogámicos C57BL , Pulmón/patología , Enfermedades Pulmonares/patología , Hemorragia , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/inducido químicamente , Lupus Eritematoso Sistémico/tratamiento farmacológico , Vasculitis/patología , Estrés del Retículo EndoplásmicoRESUMEN
PURPOSE: Common variable immunodeficiency (CVID) is the most prevalent symptomatic immunodeficiency in adults. Little is known about the manifestations of CVID presenting in older adults. Herein, we performed a phenotypic characterization of patients diagnosed older than age 40. METHODS: A retrospective chart review of 79 patients seen at UF Health between 2006 and 2020 with a verified diagnosis of CVID per the ICON 2016 criteria was conducted. Patients were classified according to four phenotypes: no-disease-related complications, autoimmune cytopenias, polyclonal lymphoproliferation, and unexplained enteropathy. Patients diagnosed with CVID from age 2 to 40 (n = 41, "younger cohort") were compared to patients diagnosed with CVID age 41 and older (n = 38, "older cohort"). RESULTS: Among the younger cohort, pathologic genetic variants, positive family history for immunodeficiency, autoimmunity (49% vs 24%, p = 0.03), and splenomegaly (46% vs 16%, p = 0.004) were more common, as was the "autoimmune cytopenias" phenotype (24% vs 3%, p = 0.007). Among the older cohort, lymphoma (11% vs 0%, p = 0.049) and the "no disease-related complications" phenotype (79% vs 57%, p = 0.03) were more commonly seen. Comorbidities such as bronchiectasis (27% vs 21%, p = 0.61), GI involvement (34% vs 24%, p = 0.33), and GLILD (5% vs 8%, p = 0.67) were equally present among both the younger and older cohorts, respectively. CONCLUSION: The lower incidence of autoimmunity and splenomegaly, as well as overlapping clinical features with immunosenescence, may make diagnosing CVID in older patients more challenging; however, the disease is not more indolent as the risks for lymphoma, bronchiectasis, and GLILD are similar to those of younger patients.
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Bronquiectasia , Inmunodeficiencia Variable Común , Leucopenia , Bronquiectasia/diagnóstico , Bronquiectasia/epidemiología , Bronquiectasia/etiología , Inmunodeficiencia Variable Común/diagnóstico , Inmunodeficiencia Variable Común/epidemiología , Inmunodeficiencia Variable Común/genética , Humanos , Fenotipo , Estudios Retrospectivos , Esplenomegalia/complicaciones , Esplenomegalia/etiologíaRESUMEN
C57BL/6 mice with pristane-induced lupus develop macrophage-dependent diffuse alveolar hemorrhage (DAH), which is blocked by treatment with liver X receptor (LXR) agonists and is exacerbated by low IL-10 levels. Serp-1, a myxomavirus-encoded serpin that impairs macrophage activation and plasminogen activation, blocks DAH caused by MHV68 infection. We investigated whether Serp-1 also could block DAH in pristane-induced lupus. Pristane-induced DAH was prevented by treatment with recombinant Serp-1 and macrophages from Serp1-treated mice exhibited an anti-inflammatory M2-like phenotype. Therapy activated LXR, promoting M2 polarization and expression of Kruppel-like factor-4 (KLH4), which upregulates IL-10. In contrast, deficiency of tissue plasminogen activator or plasminogen activator inhibitor had little effect on DAH. We conclude that Serp-1 blocks pristane-induced lung hemorrhage by enhancing LXR-regulated M2 macrophage polarization and KLH4-regulated IL-10 production. In view of the similarities between DAH in pristane-treated mice and SLE patients, Serp-1 may represent a potential new therapy for this severe complication of SLE.
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Lupus Eritematoso Sistémico/terapia , Macrófagos/efectos de los fármacos , Serpinas/farmacología , Proteínas Virales/farmacología , Animales , Coagulación Sanguínea , Femenino , Hemorragia/sangre , Hemorragia/patología , Hemorragia/prevención & control , Interleucina-10/biosíntesis , Factor 4 Similar a Kruppel , Receptores X del Hígado/metabolismo , Enfermedades Pulmonares/sangre , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/prevención & control , Lupus Eritematoso Sistémico/inducido químicamente , Lupus Eritematoso Sistémico/inmunología , Macrófagos/clasificación , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Myxoma virus/genética , Células RAW 264.7 , Serpinas/genética , Terpenos/toxicidad , Proteínas Virales/genéticaAsunto(s)
Glomerulonefritis , Gripe Humana , Enfermedades Pulmonares , Poliangitis Microscópica , Glomerulonefritis/complicaciones , Glomerulonefritis/diagnóstico , Hemorragia/diagnóstico , Hemorragia/etiología , Humanos , Gripe Humana/complicaciones , Gripe Humana/diagnóstico , Enfermedades Pulmonares/diagnóstico , Enfermedades Pulmonares/etiología , Poliangitis Microscópica/complicaciones , Poliangitis Microscópica/diagnósticoRESUMEN
OBJECTIVE: Pristane-induced lupus is associated with nonresolving inflammation and deficiency of proresolving macrophages. Proresolving nonclassic macrophages (NCMs) are less responsive to type I interferon (IFN) than classic macrophages (CMs; which are proinflammatory), reflecting their relative expression levels of the type I IFN receptor (IFNAR). This study was undertaken to investigate the regulation of IFNAR expression in macrophages. METHODS: We carried out gene expression profiling of purified CMs and NCMs from mice treated with pristane (which develop lupus) or mineral oil (non-lupus controls). Macrophage differentiation and IFNAR expression were examined in mice treated with NF-E2-related factor 2 (Nrf2) activators and inhibitors and in Nrf2-deficient mice. Nrf2 activity was also assessed in blood cells from patients with systemic lupus erythematosus (SLE). Significant differences were determined by Student's t-test. RESULTS: RNA sequencing revealed increased expression of genes regulated by the transcription factor Nrf2 in NCMs from mineral oil-treated versus pristane-treated mice and in NCMs versus CMs. The Nrf2 activator CDDO-imidazole (CDDO-Im) decreased CMs (P < 0.0001) and promoted the development of proresolving NCMs (P = 0.06), whereas the Nrf2 inhibitor brusatol increased CMs (P < 0.05) and decreased NCMs (P < 0.001). CDDO-Im decreased Ifnar1 (P < 0.001) and IFN-stimulated gene (ISG) expression in macrophages and alleviated oxidative stress (P < 0.05), whereas brusatol had the opposite effect (P < 0.01). Moreover, Ifnar1 and ISG expression levels were higher in Nrf2-knockout mice than controls (P < 0.05). As seen in mice with lupus, SLE patients showed evidence of low Nrf2 activity. CONCLUSION: Our findings indicate that Nrf2 activation favors the resolution of chronic inflammation in lupus. Since autoantibody production and lupus nephritis depend on IFNAR signaling, the ability of Nrf2 activators to repolarize macrophages and reduce the INF signature suggests that these agents may warrant consideration for treating lupus.
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Polaridad Celular/efectos de los fármacos , Lupus Eritematoso Sistémico/metabolismo , Macrófagos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Receptores de Interferón/metabolismo , Animales , Expresión Génica , Imidazoles/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacología , Cuassinas/farmacologíaRESUMEN
OBJECTIVE: Peripheral blood mononuclear cells (PBMCs) in systemic lupus erythematosus (SLE) patients exhibit a gene expression program (interferon [IFN] signature) that is attributed to overproduction of type I IFNs by plasmacytoid dendritic cells. Type I IFNs have been thought to play a role in the pathogenesis of SLE. This study was undertaken to examine an unexpected influence of monocyte/macrophages on the IFN signature. METHODS: Proinflammatory (classic) and antiinflammatory (nonclassic) monocyte/macrophages were sorted from mice and analyzed by RNA sequencing and quantitative polymerase chain reaction (qPCR). Type I IFN-α/ß/ω receptor (IFNAR-1) expression was determined by qPCR and flow cytometry. Macrophages were stimulated in vitro with IFNα, and pSTAT1was measured. RESULTS: Transcriptional profiling of peritoneal macrophages from mice with pristane-induced SLE unexpectedly indicated a strong IFN signature in classic, but not nonclassic, monocyte/macrophages exposed to the same type I IFN concentrations. Ifnar1 messenger RNA and IFNAR surface staining were higher in classic monocyte/macrophages versus nonclassic monocyte/macrophages (P < 0.0001 and P < 0.05, respectively, by Student's t-test). Nonclassic monocyte/macrophages were also relatively insensitive to IFNα-driven STAT1 phosphorylation. Humans exhibited a similar pattern: higher IFNAR expression (P < 0.0001 by Student's t-test) and IFNα-stimulated gene expression (P < 0.01 by paired Wilcoxon's rank sum test) in classic monocyte/macrophages and lower levels in nonclassic monocyte/macrophages. CONCLUSION: This study revealed that the relative abundance of different monocyte/macrophage subsets helps determine the magnitude of the IFN signature. Responsiveness to IFNα signaling reflects differences in IFNAR expression in classic (high IFNAR) compared to nonclassic (low IFNAR) monocyte/macrophages. Thus, the IFN signature depends on both type I IFN production and the responsiveness of monocyte/macrophages to IFNAR signaling.
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Factores Inmunológicos/farmacología , Interferón-alfa/farmacología , Lupus Eritematoso Sistémico/inmunología , Macrófagos Peritoneales/efectos de los fármacos , Monocitos/efectos de los fármacos , Factor de Transcripción STAT1/efectos de los fármacos , Animales , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Monocitos/inmunología , Monocitos/metabolismo , Fosforilación/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Receptor de Interferón alfa y beta/metabolismo , Factor de Transcripción STAT1/metabolismo , Análisis de Secuencia de ARNRESUMEN
Diffuse alveolar hemorrhage (DAH) is a life-threatening pulmonary complication associated with systemic lupus erythematosus, vasculitis, and stem cell transplant. Little is known about the pathophysiology of DAH, and no targeted therapy is currently available. Pristane treatment in mice induces systemic autoimmunity and lung hemorrhage that recapitulates hallmark pathologic features of human DAH. Using this experimental model, we performed high-dimensional analysis of lung immune cells in DAH by mass cytometry and single-cell RNA sequencing. We found a large influx of myeloid cells to the lungs in DAH and defined the gene expression profile of infiltrating monocytes. Bone marrow-derived inflammatory monocytes actively migrated to the lungs and homed adjacent to blood vessels. Using 3 models of monocyte deficiency and complementary transfer studies, we established a central role of inflammatory monocytes in the development of DAH. We further found that the myeloid transcription factor interferon regulatory factor 8 is essential to the development of both DAH and type I interferon-dependent autoimmunity. These findings collectively reveal monocytes as a potential treatment target in DAH.
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Hemorragia/inmunología , Enfermedades Pulmonares/inmunología , Monocitos/inmunología , Alveolos Pulmonares/patología , Animales , Separación Celular , Femenino , Citometría de Flujo , Hemorragia/patología , Humanos , Enfermedades Pulmonares/patología , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/inmunología , Masculino , Ratones , Ratones Noqueados , Monocitos/metabolismo , Alveolos Pulmonares/inmunología , RNA-Seq , Análisis de la Célula Individual , Trasplante de Células Madre/efectos adversosRESUMEN
BACKGROUND: The aim of this study was to explore the effects of irisin on human visceral adipose tissue and adipocytes functions. METHODS: Fresh human visceral white adipose tissues derived from 11 donors were used to examine the effects of irisin on browning, adipogenesis and osteogenesis gene expression, and anti-inflammatory properties. Preadipocytes were also used to examine the effects of irisin on mitochondrial respiration, adipogenic differentiation, and osteogenic differentiation. KEY RESULTS: Irisin significantly increased cellular mitochondrial energy metabolism in differentiated visceral adipocytes. Irisin also increased mRNA levels of transcriptional regulators of brite/beige adipocytes (UCP-1, PGC1α, PRDM16, TMEM26, and CD137) in subcutaneous white adipose tissue but not in visceral/brown adipose tissue or their derived mature adipocytes. In parallel, irisin increased the protein levels of UCP-1 in subcutaneous white adipose tissue, but had no effect on the expression of this protein in visceral white adipose tissue and perirenal brown adipose tissue. However, irisin inhibited adipogenic differentiation, promoted osteogenic differentiation in visceral adipocytes, down-regulated adipogenesis, and upregulated osteogenesis genes expression in visceral fat tissue. Moreover, administration of irisin reduced the expression of proinflammatory marker mRNAs in both visceral and subcutaneous white adipose tissue. CONCLUSIONS: Our data suggest that (1) irisin may increase mitochondrial respiration and glycolysis in visceral adipocytes by a UCP-1 independent pathway; (2) irisin promotes anti-inflammatory activity on fat tissue.
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The generation of CD138+ phagocytic macrophages with an alternative (M2) phenotype that clear apoptotic cells from tissues is defective in lupus. Liver X receptor-alpha (LXRα) is an oxysterol-regulated transcription factor that promotes reverse cholesterol transport and alternative (M2) macrophage activation. Conversely, hypoxia-inducible factor 1-α (HIF1α) promotes classical (M1) macrophage activation. The objective of this study was to see if lupus can be treated by enhancing the generation of M2-like macrophages using LXR agonists. Peritoneal macrophages from pristane-treated mice had an M1 phenotype, high HIFα-regulated phosphofructokinase and TNFα expression (quantitative PCR, flow cytometry), and low expression of the LXRα-regulated gene ATP binding cassette subfamily A member 1 (Abca1) and Il10 vs. mice treated with mineral oil, a control inflammatory oil that does not cause lupus. Glycolytic metabolism (extracellular flux assays) and Hif1a expression were higher in pristane-treated mice (M1-like) whereas oxidative metabolism and LXRα expression were higher in mineral oil-treated mice (M2-like). Similarly, lupus patients' monocytes exhibited low LXRα/ABCA1 and high HIF1α vs. CONTROLS: The LXR agonist T0901317 inhibited type I interferon and increased ABCA1 in lupus patients' monocytes and in murine peritoneal macrophages. In vivo, T0901317 induced M2-like macrophage polarization and protected mice from diffuse alveolar hemorrhage (DAH), an often fatal complication of lupus. We conclude that end-organ damage (DAH) in murine lupus can be prevented using an LXR agonist to correct a macrophage differentiation abnormality characteristic of lupus. LXR agonists also decrease inflammatory cytokine production by human lupus monocytes, suggesting that these agents may be have a role in the pharmacotherapy of lupus.
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Hidrocarburos Fluorados/farmacología , Receptores X del Hígado/agonistas , Macrófagos Peritoneales/efectos de los fármacos , Sulfonamidas/farmacología , Animales , Polaridad Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Hemorragia/prevención & control , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/metabolismo , Macrófagos Peritoneales/patología , Macrófagos Peritoneales/fisiología , Ratones , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Terpenos/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a "first-in-class" therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next-generation RCL peptides with improved therapeutic potential that is not reduced when the peptides are combined with antibiotic treatments. The crystal structure of cleaved Serp-1 was determined to 2.5 Å resolution and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and inhibit mammalian serpins, plasminogen activator inhibitor 1 (PAI-1), anti-thrombin III (ATIII), and α-1 antitrypsin (A1AT), and target proteases. Using in silico modeling of the Serp-1 RCL peptide, S-7, we designed several modified RCL peptides that were predicted to have stronger interactions with human serpins because of the larger number of stabilizing hydrogen bonds. Two of these peptides (MPS7-8 and -9) displayed extended activity, improving survival where activity was previously lost in antibiotic-treated MHV68-infected mice (P < 0.0001). Mass spectrometry and kinetic assays suggest interaction of the peptides with ATIII, A1AT, and target proteases in mouse and human plasma. In summary, we present the next step toward the development of a promising new class of anti-inflammatory serpin-based therapeutics.
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Factores Inmunológicos/química , Myxoma virus/química , Péptidos/química , Serpinas/química , Proteínas Virales/química , Animales , Células CHO , Cricetulus , Cristalografía por Rayos X , Humanos , Factores Inmunológicos/farmacología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Péptidos/farmacología , Infecciones por Poxviridae/virología , Conformación Proteica , Conejos , Serpinas/farmacología , Proteínas Virales/farmacologíaAsunto(s)
Riñón , Ácido Micofenólico/administración & dosificación , Nefritis Intersticial , Síndrome de Sjögren , Adulto , Antibióticos Antineoplásicos/administración & dosificación , Biopsia/métodos , Femenino , Humanos , Riñón/patología , Riñón/fisiopatología , Pruebas de Función Renal/métodos , Nefritis Intersticial/diagnóstico , Nefritis Intersticial/tratamiento farmacológico , Nefritis Intersticial/etiología , Nefritis Intersticial/fisiopatología , Síndrome de Sjögren/diagnóstico , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/fisiopatología , Síndrome de Sjögren/terapia , Resultado del TratamientoRESUMEN
Dead cells accumulating in the tissues may contribute to chronic inflammation. We examined the cause of impaired apoptotic cell clearance in human and murine lupus. Dead cells accumulated in bone marrow from lupus patients but not from nonautoimmune patients undergoing myeloablation, where they were efficiently removed by macrophages (MΦ). Impaired apoptotic cell uptake by MΦ also was seen in mice treated i.p. with pristane (develop lupus) but not mineral oil (MO) (do not develop lupus). The inflammatory response to both pristane and MO rapidly depleted resident (Tim4+) large peritoneal MΦ. The peritoneal exudate of pristane-treated mice contained mainly Ly6Chi inflammatory monocytes; whereas in MO-treated mice, it consisted predominantly of a novel subset of highly phagocytic MΦ resembling small peritoneal MΦ (SPM) that expressed CD138+ and the scavenger receptor Marco. Treatment with anti-Marco-neutralizing Abs and the class A scavenger receptor antagonist polyinosinic acid inhibited phagocytosis of apoptotic cells by CD138+ MΦ. CD138+ MΦ expressed IL-10R, CD206, and CCR2 but little TNF-α or CX3CR1. They also expressed high levels of activated CREB, a transcription factor implicated in generating alternatively activated MΦ. Similar cells were identified in the spleen and lung of MO-treated mice and also were induced by LPS. We conclude that highly phagocytic, CD138+ SPM-like cells with an anti-inflammatory phenotype may promote the resolution of inflammation in lupus and infectious diseases. These SPM-like cells are not restricted to the peritoneum and may help clear apoptotic cells from tissues such as the lung, helping to prevent chronic inflammation.
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Lupus Eritematoso Sistémico/inmunología , Macrófagos Peritoneales/inmunología , Fagocitosis , Sindecano-1/inmunología , Animales , Antígenos Ly/análisis , Apoptosis , Células de la Médula Ósea/inmunología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Humanos , Inflamación/inmunología , Subunidad alfa del Receptor de Interleucina-10/genética , Subunidad alfa del Receptor de Interleucina-10/inmunología , Pulmón/citología , Pulmón/inmunología , Lupus Eritematoso Sistémico/inducido químicamente , Lupus Eritematoso Sistémico/fisiopatología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Aceite Mineral/farmacología , Poli I/farmacología , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Bazo/citología , Bazo/inmunología , Sindecano-1/genética , Terpenos/farmacología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: Diffuse alveolar hemorrhage (DAH) in lupus patients confers >50% mortality, and the cause is unknown. We undertook this study to examine the pathogenesis of DAH in C57BL/6 mice with pristane-induced lupus, a model of human lupus-associated DAH. METHODS: Clinical/pathologic and immunologic manifestations of DAH in pristane-induced lupus were compared with those of DAH in humans. Tissue distribution of pristane was examined by mass spectrometry. Cell types responsible for disease were determined by in vivo depletion using clodronate liposomes and antineutrophil monoclonal antibodies (anti-Ly-6G). The effect of complement depletion with cobra venom factor (CVF) was examined. RESULTS: After intraperitoneal injection, pristane migrated to the lung, causing cell death, small vessel vasculitis, and alveolar hemorrhage similar to that seen in DAH in humans. B cell-deficient mice were resistant to induction of DAH, but susceptibility was restored by infusing IgM. C3-/- and CD18-/- mice were also resistant, and DAH was prevented in wild-type mice by CVF. Induction of DAH was independent of Toll-like receptors, inflammasomes, and inducible nitric oxide. Mortality was increased in interleukin-10 (IL-10)-deficient mice, and pristane treatment decreased IL-10 receptor expression in monocytes and STAT-3 phosphorylation in lung macrophages. In vivo neutrophil depletion was not protective, while treatment with clodronate liposomes prevented DAH, which suggests that macrophage activation is central to DAH pathogenesis. CONCLUSION: The pathogenesis of DAH involves opsonization of dead cells by natural IgM and complement followed by complement receptor-mediated lung inflammation. The disease is macrophage dependent, and IL-10 is protective. Complement inhibition and/or macrophage-targeted therapies may reduce mortality in lupus-associated DAH.
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Hemorragia/etiología , Enfermedades Pulmonares/etiología , Lupus Eritematoso Sistémico/complicaciones , Animales , Modelos Animales de Enfermedad , Femenino , Hemorragia/patología , Humanos , Interleucina-10/metabolismo , Pulmón/patología , Enfermedades Pulmonares/patología , Lupus Eritematoso Sistémico/inducido químicamente , Lupus Eritematoso Sistémico/patología , Activación de Macrófagos/fisiología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Alveolos Pulmonares/patología , TerpenosRESUMEN
The unusual subset of patients with severe hepatitis, hypergammaglobulinemia, arthritis, and LE cells in the blood reported by Henry Kunkel and others suggested to these investigators that "lupoid" hepatitis might share pathogenic mechanisms with SLE. More than half a century later, the etiology of autoimmune hepatitis remains unclear. The occurrence of autoimmune hepatitis in a small fraction (about 3%) of SLE patients in our lupus cohort and in two mouse models of SLE supports their conclusion that lupoid hepatitis may be share pathogenic mechanisms with SLE. The development of autoimmune hepatitis in mice with pristane-induced lupus provides an opportunity to further explore the potential link between these two autoimmune disorders.
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Hepatitis Autoinmune , Lupus Eritematoso Sistémico , Actinas/inmunología , Adulto , Anciano , Animales , Autoanticuerpos/sangre , Modelos Animales de Enfermedad , Femenino , Hepatitis Autoinmune/sangre , Hepatitis Autoinmune/inmunología , Hepatitis Autoinmune/patología , Humanos , Inmunoglobulina G/sangre , Hígado/patología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inducido químicamente , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Persona de Mediana Edad , Músculo Liso/inmunología , Seroglobulinas/análisis , Terpenos , Adulto JovenRESUMEN
Anti-muscarinic type 3 receptor autoantibodies (anti-M3R) are reported as potential inhibitors of saliva secretion in Sjögren's syndrome (SjS). However, despite extensive efforts to establish an anti-M3R detection method, there is no clinical test available for these autoantibodies. The purpose of this study was to propose inclusion of anti-M3R testing for SjS diagnosis through investigation of their prevalence using a modified In-Cell Western (ICW) assay. A stable cell line expressing human M3R tagged with GFP (M3R-GFP) was established to screen unadsorbed and adsorbed plasma from primary SjS (n=24), rheumatoid arthritis (RA, n=18), systemic lupus erythematosus (SLE, n=18), and healthy controls (HC, n=23). Anti-M3R abundance was determined by screening for the intensity of human IgG interacting with M3R-GFP cells by ICW assay, as detected by an anti-human IgG IRDye800-conjugated secondary antibody and normalized to GFP. Method comparisons and receiver-operating-characteristic (ROC)-curve analyses were performed to evaluate the diagnostic value of our current approaches. Furthermore, clinical parameters of SjS were also analyzed in association with anti-M3R. Anti-M3R was significantly elevated in SjS plasma in comparison with HC, SLE, or RA (P<0.01). SjS anti-M3R intensities were greater than two-standard deviations above the HC mean for both unadsorbed (16/24, 66.67%) and adsorbed (18/24, 75%) plasma samples. Furthermore, anti-M3R was associated with anti-SjS-related-antigen A/Ro positivity (P=0.0353). Linear associations for anti-M3R intensity indicated positive associations with focus score (R(2)=0.7186, P<0.01) and negative associations with saliva flow rate (R(2)=0.3052, P<0.05). Our study strongly supports our rationale to propose inclusion of anti-M3R for further testing as a non-invasive serological marker for SjS diagnosis.