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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.

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.

Chronic peritoneal inflammation following pristane injection induces lupus with diffuse alveolar hemorrhage (DAH) and pulmonary capillaritis in C57BL/6 mice. The pathogenesis involves pristane-induced microvascular lung injury. BALB/c mice are resistant to endothelial injury and DAH. Lung disease in C57BL/6 mice is abolished by depleting monocytes/macrophages. The objective of this study was to define the role of myeloid subsets in DAH. Hemorrhage and vasculitis were abolished in Ccr2-/- mice, indicating involvement of bone marrow-derived monocytes/macrophages. Along with Ly6Chi monocytes, we found two subsets of circulating Ly6Clo monocytes: one CD138- and a novel CD138+ subset. Nr4a1-dependent patrolling Ly6Clo monocytes maintain vascular integrity after endothelial injury. Circulating Ly6CloCD138+ monocytes were associated with DAH and were absent in mice without DAH. They also were absent in Nr4a1-/- mice, whereas Ly6CloCD138- monocytes were unaffected. However, Nr4a1-/- mice were susceptible to pristane-induced DAH and lung vasculitis, suggesting that disease onset does not require Ly6CloCD138- monocytes. Peritoneal Ly6CloCD138+ Mϕ were unchanged in Nr4a1-/- mice, indicating that they are not derived from Ly6CloCD138+ monocytes. We conclude that pristane-induced lung microvascular lung injury stimulates a wave of Nr4h1-dependent Ly6CloCD138+ patrolling monocytes in an ineffectual effort to maintain vascular integrity in the face of ongoing endothelial damage.

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/β/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.

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+ cytotoxic T lymphocytes (CTL). However, the molecular mechanisms impacting CTL function remain unknown. Here, we studied the role of NOX2 in both non-obese diabetic (NOD) mouse and human CTL function. Genetic ablation or chemical inhibition of NOX2 in CTL significantly suppressed activation-induced expression of the transcription factor T-bet, the master transcription factor of the Tc1 cell lineage, and T-bet target effectors genes such as IFNγ and granzyme B. Inhibition of NOX2 in both human and mouse CTL prevented target cell lysis. We identified that superoxide generated by NOX2 must be converted into hydrogen peroxide to transduce the redox signal in CTL. Further, we show that NOX2-generated oxidants deactivate the Tumor Suppressor Complex leading to mTOR complex 1 activation and CTL effector function. These results indicate that NOX2 plays a non-redundant role in T cell receptor-mediated CTL effector function.

Sjogren’s syndrome (SS) is a progressive systemic autoimmune disease causing chronic inflammation of the salivary, lacrimal, and other glands and B cell hyperactivity leading to the production of autoantibodies, such as anti-Ro (SS-A), La (SS-B), and rheumatoid factor (RF), and a 40-fold increased risk of extranodal B cell lymphoma arising in the inflamed salivary glands, especially the parotid glands. Although most of the cells infiltrating the salivary glands are T cells, B cells and plasma cells within the inflammatory infiltrates may undergo benign or malignant expansions. A variety of B cell lymphoproliferative processes are seen, both benign (e.g., lymphocytic interstitial pneumonitis) and malignant (e.g., marginal zone lymphoma, diffuse large B cell lymphoma, and follicular lymphoma). Many of the B cell lymphomas produce RF with restricted usage of somatically mutated heavy-chain genes. We discuss the pathogenesis of B cell lymphomas in SS as well as their higher frequency in primary compared with secondary SS. Finally, we review clinical aspects of lymphoid neogenesis in SS, including the risk factors, diagnostic considerations, and therapeutic options.

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.

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.

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.

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.

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.

Background Peripheral blood cells from two-thirds of adult lupus patients exhibit a gene expression program (interferon signature) attributed to the over-production of interferon (IFN) and other type I IFNs (IFN-I). IFN-I may be involved in the pathogenesis of lupus. Although plasmacytoid dendritic cells produce large amounts of IFN-I, our studies in an experimental lupus model suggest that macrophages and/or monocytes may play an important role in generating the interferon signature. This study sought to define how macrophages influence the interferon signature. Methods Proinflammatory and anti-inflammatory (pro-resolving) macrophages were isolated from mice with pristane-induced lupus and were analyzed by RNA-sequencing (RNA-Seq) and quantitative PCR (qPCR). Protein expression in macrophage subsets was evaluated by flow cytometry. The role of nuclear factor erythroid 2 like 2 (Nrf2) activators was examined in vivo Results Transcriptional profiling (RNA-Seq) of CD11b+Ly6G- peritoneal macrophages from mice with experimental lupus unexpectedly indicated a strong interferon signature in proinflammatory (Ly6ChiCD138-), but not anti-inflammatory (Ly6C-CD138+), macrophages exposed to the same IFN-I concentrations. Along with higher IFN-I regulated gene expression, proinflammatory macrophages expressed lower levels of genes regulated by nuclear factor erythroid 2 like 2 (Nrf2) than anti-inflammatory macrophages. Transcript levels of IFN receptor 1 chain (Ifnar1), IFNAR surface staining, and mitochondrial superoxide all were higher in proinflammatory macrophages. Administration of the Nrf2 activator CDDO-Im to lupus mice decreased IFNAR expression, blocked IFN-driven Stat1 phosphorylation, and reduced IFN-regulated gene expression. Thus, the interferon signature in murine lupus critically depends on Nrf2-regulated changes in IFNAR expression in macrophages. Human peripheral blood mononuclear cells exhibited a similar pattern: high IFNAR expression in classical monocytes and lower levels in non-classical monocytes. The data suggest that anti-inflammatory macrophages/monocytes are insensitive to IFN signaling, potentially serving a role in the resolution of inflammation. Conclusions These studies reveal that the relative abundance of different monocyte/macrophage subsets (proinflammatory vs. anti-inflammatory/pro-resolving) helps determine the magnitude of the interferon signature. In addition to reflecting IFN-I production, the interferon signature critically depends on the Nrf2-regulated responsiveness of macrophages to signaling through the IFNAR. These studies provide a new perspective on the interferon signature that may have implications for understanding the pathogenesis of lupus. In addition, these studies suggest that Nrf2 activators could have a role in the therapy of lupus. Funding Source(s): NIH

Objective In vitro studies suggest that the type I interferon (IFN) signature seen in most lupus patients results from Fcγ receptor–mediated uptake of nucleic acid–containing immune complexes by plasmacytoid dendritic cells and engagement of endosomal Toll-like receptors. The aim of this study was to reexamine the pathogenesis of the IFN signature in vivo. Methods Lupus was induced in mice by injecting pristane. Some mice were treated with normal immunoglobulin or with cobra venom factor to deplete complement. The IFN signature was evaluated by polymerase chain reaction. The IFN signature also was determined in C4-deficient patients and control subjects. Results Wild-type C57BL/6 mice with pristane-induced lupus developed a strong IFN signature, which was absent in immunoglobulin-deficient (μMT), C3−/−, and CD18−/− mice. Intravenous infusion of normal IgM, but not IgG, restored the IFN signature in μMT mice, and the IFN signature in wild-type mice was inhibited by depleting complement, suggesting that opsonization by IgM and complement is involved in IFN production. Consistent with that possibility, the levels of “natural” IgM antibodies reactive with dead cells were increased in pristane-treated wild-type mice compared with untreated controls, and in vivo phagocytosis of dead cells was impaired in C3-deficient mice. To examine the clinical relevance of these findings, we identified 10 C4-deficient patients with lupus-like disease and compared them with 152 C4-intact patients and 21 healthy controls. In comparison with C4-intact patients, C4-deficient patients had a different clinical/serologic phenotype and lacked the IFN signature. Conclusion These studies define previously unrecognized roles of natural IgM, complement, and complement receptors in generating the IFN signature in lupus.

Human noroviruses are a leading cause of gastroenteritis, and so, vaccine development is desperately needed. Elucidating viral mechanisms of immune antagonism can provide key insight into designing effective immunization platforms. We recently revealed that B cells are targets of norovirus infection. Because noroviruses can regulate antigen presentation by infected macrophages and B cells can function as antigen-presenting cells, we tested whether noroviruses regulate B-cell-mediated antigen presentation and the biological consequence of such regulation. Indeed, murine noroviruses could prevent B-cell expression of antigen presentation molecules and this directly correlated with impaired control of acute infection. In addition to B cells, acute control required MHC class I molecules, CD8+ T cells, and granzymes, supporting a model whereby B cells act as antigen presenting cells to activate cytotoxic CD8+ T cells. This immune pathway was active prior to the induction of antiviral antibody responses. As in macrophages, the minor structural protein VP2 regulated B-cell antigen presentation in a virus-specific manner. Commensal bacteria were not required for the activation of this pathway and ultimately only B cells were required for the clearance of viral infection. These findings provide new insight into the role of B cells in stimulating antiviral CD8+ T-cell responses.

Background Although SLE is a prototype of autoimmune (T cell/B cell-mediated) disease, there is increasing evidence implicating myeloid cells in its pathogenesis. We examined the role of macrophages (MΦ) in human SLE and the pristane-induced lupus model. Methods Tissues of SLE patients and mice with pristane-induced lupus were examined by double immunohistochemistry. MΦ were analyzed by flow cytometry, phagocytosis assay, real-time PCR, Seahorse assay, and RNA-Seq. Mice were injected daily with the liver X receptor (LXR) agonist T0901317. Results SLE patients’ bone marrow contained numerous activated caspase-3+ apoptotic cells located outside of MΦ. In contrast, in leukemia patients undergoing bone marrow ablation prior to transplantation, all caspase-3+ cells were inside of MΦ, suggesting that lupus is associated with impaired clearance of apoptotic cells. MΦ in pristane-lupus took up fluorescently-labeled apoptotic cells poorly compared with controls. Studies in pristane-treated knock-out mice indicated that the induction of diffuse alveolar hemorrhage (DAH), a serious pulmonary complication of lupus, required opsonization of dead cells in the lung by ‘natural’ IgM (DAH absent in μMT mice, restored by infusion of IgM), C3, and C3b receptors (absent in C3-/- and CD18-/- mice, prevented by complement depletion with cobra venom factor). DAH also was prevented by MΦ depletion (clodronate liposomes) but not neutrophil depletion (anti-Ly6G). MΦ in pristane-induced lupus exhibited features of classical activation (impaired phagocytosis of apoptotic cells, high glycolysis, low oxidative phosphorylation, increased HIF1α expression, M1 surface markers, and TNFα production), whereas control MΦ from mice treated with mineral oil (do not develop DAH) were M2-like, with high phagocytic activity, low glycolysis, high OxPhos, increased LXRα expression, M2 surface markers, and IL-10 production. In both pristane-induced lupus MΦ and SLE patients’ monocytes, we found low levels of the LXRα-regulated reverse cholesterol transporter ABCA1. We hypothesized that it might be possible to treat DAH by ‘re-polarizing’ MΦ using a synthetic LXR agonist. Consistent with that idea, pristane-treated mice receiving T0901317 did not develop DAH and MΦ from mice receiving T0901317 exhibited an M2-like surface phenotype and decreased TNFα production compared with controls. Conclusions Our data suggest that lupus-associated DAH is partly an autoinflammatory response caused by sluggish clearance of apoptotic cells by M1-like MΦ and that certain clinical manifestations (e.g. DAH) can be treated by repolarizing MΦ using an activator of the transcription factor LXRα. Low levels of LXR-driven ABCA1 expression also may have implications for the therapy of atherosclerosis in SLE.

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 (P0.0001). 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.

Current evidence suggests that neonatal immunity is functionally distinct from adults. Although TLR signaling through the adaptor protein, MyD88, has been shown to be critical for survival to sepsis in adults, little is known about the role of MyD88 or TRIF in neonatal sepsis. We demonstrate that TRIF−/− but not MyD88−/− neonates are highly susceptible to Escherichia coli peritonitis and bacteremia. This was associated with decreased innate immune recruitment and function. Importantly, we found that the reverse was true in adults that MyD88−/− but not TRIF−/− or wild-type adults are susceptible to E. coli peritonitis and bacteremia. In addition, we demonstrate that TRIF but not MyD88 signaling is critical for the TLR4 protective adjuvant effect we have previously demonstrated. These data suggest a differential requirement for the survival of neonates versus adults to Gram-negative infection, and that modulation of TRIF in neonates can be used to augment survival to neonatal sepsis.

Objective Tumor necrosis factor α (TNFα) antagonists are effective for treating rheumatoid arthritis and other inflammatory diseases, but their use can be complicated by the development of lupus-like phenomena. This study was undertaken to investigate the role of TNFα in a murine model of lupus. Methods Toll-like receptor 7 (TLR-7) ligand–driven lupus was induced by injection of pristane into C57BL/6 (B6) mice deficient in TNFα (TNFα−/−) or TNFα-intact B6 mice as wild-type controls. Autoantibody and type I interferon (IFN) production was measured in each group of mice, and the effects of the presence or absence of TNFα on type I IFN–producing plasmacytoid dendritic cells (PDCs), Ly-6Chigh monocytes, and TNFα-producing neutrophils were determined. Results TNFα−/− mice did not spontaneously develop autoantibodies or clinical manifestations of lupus, suggesting that TNFα deficiency alone is insufficient to cause lupus. Although the levels of type I IFN were comparable between untreated TNFα−/− and wild-type control mice, untreated TNFα−/− mice had increased circulating levels of PDCs and PDC-like cells, which enhanced the potential for production of type I IFN. When treated with pristane, TNFα−/− mice developed more severe lupus compared to pristane-treated controls, characterized by increased levels of anti-Sm/RNP autoantibodies, type I IFN, PDCs, and peritoneal inflammatory (Ly-6Chigh) monocytes. In pristane-treated TNFα−/− mice, the numbers of neutrophils, a cell type that promotes resolution of inflammation, were decreased considerably, whereas the responses of inflammatory monocytes and PDCs and the production of type I IFN were increased and prolonged. Conclusion Low levels of TNFα will increase the number of circulating PDCs in mice, thereby enhancing the potential to produce type I IFN. However, this does not necessarily lead to type I IFN production or autoimmunity unless there is concomitant exposure to endogenous TLR-7 ligands, which are released from dead cells following pristane treatment. In humans, the rate of clearance of dead cells, along with the levels of TNFα, may influence who will develop lupus following treatment with TNFα inhibitors.

The generation of CD138+ phagocytic macrophages with an alternative (M2) phenotype that clear apoptotic cells from tissues is defective in lupus. Liver X receptor-α (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 LXR α 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, an often fatal complication of lupus. We conclude that end-organ damage (diffuse alveolar hemorrhage) 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

Dead cells accumulating in the tissues may contribute to chronic inflammation. We examined the cause of impaired apoptotic cell clearance human and murine lupus. Dead cells accumulated in bone marrow from lupus patients but not from non-autoimmune patients undergoing myeloablation, where they were efficiently removed by macrophages. Impaired apoptotic cell uptake by macrophages 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 macrophages (LPM). 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 macrophages resembling small peritoneal macrophages (SPM) that expressed CD138+ and the scavenger receptor Marco. Treatment with anti-Marco neutralizing antibodies and the class A scavenger receptor antagonist polyinosinic acid inhibited phagocytosis of apoptotic cells by CD138+ macrophages. CD138+ macrophages expressed IL-10 receptor, CD206, and CCR2 but little TNFα or CX3CR1. They also expressed high levels of activated CREB, a transcription factor implicated in generating alternatively activated macrophages. Similar cells were identified in the spleen and lung of MO-treated mice and also were induced by lipopolysaccharide. 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.

The onset of autoantibodies in systemic autoimmunity can be the result of a breakdown in tolerance at multiple checkpoints. Genetic, hormonal, and immunological factors can combine with environmental influences to accelerate the onset of disease and aggravate disease outcome. Here, we describe a novel mechanism relating to the regulatory role of Neutrophil Gelatinase Associated Lipocalin (NGAL) in modulating the levels of autoantibodies in pristane induced lupus. Following a single injection of pristane intraperitoneally, NGAL expression was induced in both the serum and spleen. Furthermore, NGAL deficient mice were more susceptible to the induction of pristane stimulated autoimmunity, and displayed higher numbers of autoantibody secreting cells and increased expression of activation induced cytidine deaminase (AID) and other inflammatory mediators in the spleen. In contrast, kidney damage was milder in NGAL deficient mice, indicating that NGAL was detrimental in autoantibody mediated kidney disease. These studies indicate that NGAL plays differential roles in different tissues in the context of lupus, and suggest a previously unrecognized role for NGAL in adaptive immunity.

Objective To define the pathogenesis of bone marrow (BM) involvement in systemic lupus erythematosus (SLE). Methods Tumor necrosis factor α (TNFα) levels, cell death, and cellular damage in BM from SLE patients, controls, and mice with pristane-induced lupus were analyzed using a morphometric technique and immunohistochemistry. The pathogenesis of BM abnormalities was studied in wild-type (WT), TNFα−/−, Toll-like receptor–deficient (TLR-7−/−), interferon (IFN)–α/β/ω receptor–knockout (IFNAR−/−), and B cell–deficient (μmt) mice treated with pristane. Flow cytometry was used to examine TNFα production (by intracellular staining) and plasma cell/plasmablast development. CXCL12 expression was determined by quantitative polymerase chain reaction. Results BM from SLE patients exhibited striking death of niche and hematopoietic cells associated with TNFα overproduction. BM from mice with a type I IFN–mediated lupus syndrome induced by pristane showed similar abnormalities. TNFα was produced mainly by BM neutrophils, many with phagocytosed nuclear material (lupus erythematosus cells). TNFα production was abolished in pristane-treated TLR-7−/− and μmt mice but was restored in μmt mice by infusing normal plasma. Pristane-treated WT and IFNAR−/− mice developed anemia, BM hypocellularity, and extramedullary hematopoiesis, which were absent in TLR-7−/− and TNFα−/− mice. Additionally, the expression of CXCL12, which is produced by stromal cells and mediates homing of hematopoietic cells and plasmablasts, was decreased in BM from pristane-treated WT mice but was normal in BM from pristane-treated TNFα−/− mice. Conclusion Although autoantibodies and glomerulonephritis are type I IFN dependent, lupus-associated BM abnormalities were TLR-7 and TNFα driven but type I IFN independent, suggesting that lupus is a disorder of innate immunity in which TLR-7 activation by phagocytosed nuclei causes relentless type I IFN and TNFα production mediating glomerulonephritis and hematologic involvement, respectively.

Anemia is one of the most common hematological manifestations in SLE patients, occurring in about 50% of active cases. STAT1 is a critical signaling molecule required for the production of type-1 interferon (I-IFN), CCL2, and CXCL10, all of which are upregulated in SLE. Overexpression of STAT1 has been described to be involved in anemia in animal models. The aim of this study is to analyze how these components are involved in SLE-associated anemia.Blood samples were collected from 39 healthy donors and 101 SLE patients fulfilling ACR criteria. Samples were collected from a total of 180 visits (58 patients had 2 or more visits) of which 52 visits included a diagnosis of anemia. Healthy donors had only single visit. Total RNA, isolated from leukocytes, was analyzed by Taqman qPCR. Relative expression levels of I-IFN signature genes, chemokines, and miR-146a were determined by the ΔΔCT method. Results were correlated with clinical data and analyzed by the Wilcoxon/Kruskal-Wallis test and Fisher's exact test.Significant increases in IFN score (p0.0001), STAT1 (p0.0001), miR-146a (p0.0005), CCL2 (p = 0.0047), and CXCL10 (p = 0.017), as well as a significant decrease in pri-miR-146a (p = 0.0002), were detected in the anemic SLE patient visits (n = 52) compared to non-anemic SLE visits (n = 128). Regardless of disease activity, lupus nephritis, or race, anemic SLE patients displayed significantly elevated levels of STAT1 and miR-146a compared to non-anemic SLE patients.STAT1 and miR-146a may be upregulated during inflammation and via proinflammatory cytokines and chemokines in SLE. Prolonged upregulation of STAT1 and miR-146a appears to play an important role in anemia in SLE patients.

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.

Background Sjögren’s syndrome (SjS) monocytes have a pro-inflammatory phenotype, which may influence SjS pathogenesis. MicroRNAs (miRNAs) are small endogenously expressed molecules that can inhibit protein expression of their targeted genes and have important functions in regulating cell signaling responses. We profiled miRNAs in SjS monocytes to identify a SjS-specific miRNA profile and determine the potential roles of miRNAs in SjS pathogenesis. Methods Total RNA was extracted from healthy control (HC, n = 10), SjS (n = 18), systemic lupus erythematosus (SLE, n = 10), and rheumatoid arthritis (RA, n = 10) peripheral blood CD14+ monocytes for miRNA microarray analysis. To validate select miRNAs from the microarray analysis, the original cohort and a new cohort of monocyte RNA samples from HC (n = 9), SjS (n = 12), SLE (n = 8), and RA (n = 9) patients were evaluated by quantitative reverse transcription (RT)-PCR. Functional predictions of differentially expressed miRNAs were determined through miRNA target prediction database analyses. Statistical analyses performed included one-way analysis of variance with Bonferroni post tests, linear regression, and receiver operating characteristic curve analyses. Results MiRNAs were predominantly upregulated in SjS monocytes in comparison with controls. Quantitative RT-PCR confirmations supported co-regulation of miR-34b-3p, miR-4701-5p, miR-609, miR-300, miR-3162-3p, and miR-877-3p in SjS monocytes (13/30, 43.3 %) in comparison with SLE (1/17, 5.8 %) and RA (1/18, 5.6 %). MiRNA-target pathway predictions identified SjS-associated miRNAs appear to preferentially target the canonical TGFβ signaling pathway as opposed to pro-inflammatory interleukin-12 and Toll-like receptor/NFkB pathways. Conclusions Our results underscore a novel underlying molecular mechanism where SjS-associated miRNAs may collectively suppress TGFβ signaling as opposed to pro-inflammatory interleukin-12 and Toll-like receptor/NFκB pathways in SjS pathogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-0987-0) contains supplementary material, which is available to authorized users.

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 (R2 = 0.7186, P < 0.01) and negative associations with saliva flow rate (R2 = 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.

This chapter deals with immunological tests that are useful in the diagnosis and monitoring of systemic lupus erythematosus (SLE). We review the detection and quantification of antibodies against self-antigens (autoantibodies), measurement of complement levels, and the interferon gene expression signature.

Systemic lupus erythematosus is associated with a characteristic serological signature, including most notably anti-dsDNA and anti-Sm/RNP autoantibodies. In humans, these autoantibodies may be regulated differently, as anti-dsDNA antibodies fluctuate with disease activity, whereas anti-Sm/RNP autoantibodies tend to remain at high levels for extended periods. Disorders of B-cell activation/survival, endosomal toll-like receptor/type I interferon signaling, and clearance of apoptotic cells are associated with partial lupus-like syndromes characterized by limited disease manifestations and autoantibodies. Clinically, these syndromes are most frequently associated with evidence of glomerulonephritis, such as renal immune complex deposition and histological changes. Interestingly, the requirements for generating anti-dsDNA autoantibodies appear less stringent than those for anti-Sm/RNP, suggesting that abnormal B-cell activation, interferon production, and phagocytosis of apoptotic cells may act synergistically to produce a more diverse autoantibody profile and more widespread disease.

Genetic polymorphisms of IFN regulatory factor 5 (IRF5) are associated with an increased risk of lupus in humans. In this study, we examined the role of IRF5 in the pathogenesis of pristane-induced lupus in mice. The pathological response to pristane in IRF5−/− mice shared many features with type I IFN receptor (IFNAR)−/− and TLR7−/− mice: production of anti-Sm/RNP autoantibodies, glomerulonephritis, generation of Ly6Chi monocytes, and IFN-I production all were greatly attenuated. Lymphocyte activation following pristane injection was greatly diminished in IRF5−/− mice, and Th cell differentiation was deviated from Th1 in wild-type mice toward Th2 in IRF5−/− mice. Th cell development was skewed similarly in TLR7−/− or IFNAR−/− mice, suggesting that IRF5 alters T cell activation and differentiation by affecting cytokine production. Indeed, production of IFN-I, IL-12, and IL-23 in response to pristane was markedly decreased, whereas IL-4 increased. Unexpectedly, plasmacytoid dendritic cells (pDC) were not recruited to the site of inflammation in IRF5−/− or MyD88−/− mice, but were recruited normally in IFNAR−/− and TLR7−/− mice. In striking contrast to wild-type mice, pristane did not stimulate local expression of CCL19 and CCL21 in IRF5−/− mice, suggesting that IRF5 regulates chemokine-mediated pDC migration independently of its effects on IFN-I. Collectively, these data indicate that altered production of IFN-I and other cytokines in IRF5−/− mice prevents pristane from inducing lupus pathology by broadly affecting T and B lymphocyte activation/differentiation. Additionally, we uncovered a new, IFN-I–independent role of IRF5 in regulating chemokines involved in the homing of pDCs and certain lymphocyte subsets.

In vitro studies suggest that the type I interferon (IFN) signature seen in most lupus patients results from Fcγ receptor-mediated uptake of nucleic acid-containing immune complexes by plasmacytoid dendritic cells and engagement of endosomal Toll-like receptors. The aim of this study was to reexamine the pathogenesis of the IFN signature in vivo.Lupus was induced in mice by injecting pristane. Some mice were treated with normal immunoglobulin or with cobra venom factor to deplete complement. The IFN signature was evaluated by polymerase chain reaction. The IFN signature also was determined in C4-deficient patients and control subjects.Wild-type C57BL/6 mice with pristane-induced lupus developed a strong IFN signature, which was absent in immunoglobulin-deficient (μMT), C3These studies define previously unrecognized roles of natural IgM, complement, and complement receptors in generating the IFN signature in lupus.

Anti-RNA polymerase III (RNAP III) antibodies are highly specific for scleroderma (SSc) and associated with diffuse SSc and renal crisis. Coexistence of anti-RNAP III and other SSc autoantibodies is rarely documented. We report three cases with coexisting anti-RNAP III and anti-U1RNP. Autoantibodies in 3829 sera from rheumatology clinics were screened by immunoprecipitation. Anti-RNAP III-positive sera were also examined by immunofluorescence and anti-RNAP III ELISA. In total, 35 anti-RNAP III-positive sera were identified by immunoprecipitation, in which three had coexisting anti-U1RNP. All three were anti-RNAP III ELISA positive. Two had anti-RNAP I dominant (vs. RNAP III) reactivity and showed strong nucleolar staining. A case with anti-U1/U2RNP (U2RNP dominant) had systemic lupus erythematosus (SLE)–SSc overlap syndrome; however, the remaining two cases had SLE without signs of SSc. All three cases of anti-RNAP III + U1RNP fulfilled ACR SLE criteria but none in the group with anti-RNAP III alone ( p = 0.0002). In contrast, only one case in the former group had sclerodermatous skin changes and Raynaud's phenomenon, vs. 92% with scleroderma in the latter ( p

Diffuse alveolar hemorrhage is an uncommon, yet often fatal, complication of systemic lupus erythematosus (SLE). Advances in the treatment of alveolar hemorrhage have been hampered because of the heterogeneity of clinical findings and the lack of suitable animal models. A single intraperitoneal injection of pristane induces a lupus-like syndrome characterized by lupus-related autoantibodies and glomerulonephritis in non-autoimmune-prone strains of mice. In addition, C57BL/6 (B6) mice frequently develop alveolar hemorrhage within a few weeks of pristane injection. Immunopathogenesis of pristane-induced alveolar hemorrhage was investigated in the present study. Early (2-4 weeks after injection) mortality due to hemorrhage was unique to C57BL/6 and C57BL/10 strains of mice. Recruitment of the macrophages and neutrophils preceded the hemorrhage by several days, and hemorrhage started 3-7 days after pristane injection in some mice, peaked at 2 weeks (84% in B6) and then resolved by 4 weeks in a majority of mice. Alveolar hemorrhage was independent of MyD88 (myeloid differentiation factor 88), or TLR7 pathways, in contrast to autoantibody production and glomerulonephritis, and was also independent of FcγR or Fas. Rag1(-/-) mice had a reduced prevalence of alveolar hemorrhage compared with B6 (P=0.01) congenics. However, T-cell receptor-deficient mice developed alveolar hemorrhage at a rate comparable to wild-type controls, whereas B6 Igμ(-/-) mice surprisingly had a strikingly reduced prevalence (7% vs 84% in B6, P