PROTAC-mediated NR4A1 degradation as a novel strategy for cancer immunotherapy.

An effective cancer therapy requires killing cancer cells and targeting the tumor microenvironment (TME). Searching for molecules critical for multiple cell types in the TME, we identified NR4A1 as one such molecule that can maintain the immune suppressive TME. Here, we establish NR4A1 as a valid target for cancer immunotherapy and describe a first-of-its-kind proteolysis-targeting chimera (PROTAC, named NR-V04) against NR4A1. NR-V04 degrades NR4A1 within hours in vitro and exhibits long-lasting NR4A1 degradation in tumors with an excellent safety profile. NR-V04 inhibits and frequently eradicates established tumors. At the mechanistic level, NR-V04 induces the tumor-infiltrating (TI) B cells and effector memory CD8+ T (Tem) cells and reduces monocytic myeloid-derived suppressor cells (m-MDSC), all of which are known to be clinically relevant immune cell populations in human melanomas. Overall, NR-V04-mediated NR4A1 degradation holds promise for enhancing anticancer immune responses and offers a new avenue for treating various types of cancers such as melanoma.

A novel monocyte differentiation pattern in pristane-induced lupus with diffuse alveolar hemorrhage.

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.

Microvascular lung injury and endoplasmic reticulum stress in systemic lupus erythematosus-associated alveolar hemorrhage and pulmonary vasculitis.

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.

CD177 modulates the function and homeostasis of tumor-infiltrating regulatory T cells.

Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177+ TI Treg population that may serve as a target for TI Treg-specific immunotherapy.

Myxomavirus serpin alters macrophage function and prevents diffuse alveolar hemorrhage in pristane-induced lupus.

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.

NF-E2-Related Factor 2 Regulates Interferon Receptor Expression and Alters Macrophage Polarization in Lupus.

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.

Differential Responsiveness of Monocyte and Macrophage Subsets to Interferon.

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.

High-dimensional analysis reveals a pathogenic role of inflammatory monocytes in experimental diffuse alveolar hemorrhage.

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.

Liver X Receptor Agonist Therapy Prevents Diffuse Alveolar Hemorrhage in Murine Lupus by Repolarizing Macrophages.

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.

A Novel Subset of Anti-Inflammatory CD138+ Macrophages Is Deficient in Mice with Experimental 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.

Pathogenesis of Diffuse Alveolar Hemorrhage in Murine Lupus.

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.

Mechanisms of tumor necrosis factor α antagonist-induced lupus in a murine model.

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-6C(high) 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-6C(high) ) 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.

Maintenance of autoantibody production in pristane-induced murine lupus.

BACKGROUND: Pristane-treated mice chronically produce high levels of anti-ribonucleoprotein/Smith (anti-Sm/RNP) and other lupus autoantibodies. The present study addressed how these autoantibody levels are maintained over time. METHODS: Lupus was induced in BALB/c mice using pristane. Naïve B cells, switched memory B cells, switched plasmablasts, and plasma cells were flow-sorted and total IgG and anti-U1A (RNP) autoantibodies were determined with ELISA. RESULTS: B cells with a switched "memory-like" (CD19(+)CD138(-)IgM(-)IgD(-)) (sMB) phenotype were increased in pristane-treated mice and expressed higher levels of Toll like receptor 7 (Tlr7) than cells with this phenotype from untreated mice. Flow-sorted sMB cells from pristane-treated mice did not secrete IgG spontaneously, but were hyper-responsive to both synthetic (R848) and natural (apoptotic cells) TLR7 ligands, resulting in increased IgG production in vitro. The flow-sorted sMB cells also could be driven by R848 to produce IgG anti-U1A autoantibodies. Production of IgG was strongly inhibited by both JSH-23 and SB203580, suggesting that the canonical NFκB and p38 MAPK pathways, respectively, contribute to the TLR7 ligand hyper-responsiveness of sMB from pristane-treated mice. CONCLUSIONS: The switched memory B cell subset from pristane-treated mice is expanded and shows an increased propensity to undergo terminal (plasma cell) differentiation in response to synthetic and natural TLR7 ligands. The data suggest that the decreased clearance of apoptotic cells characteristic of pristane-treated mice might help maintain high serum levels of anti-RNP/Sm autoantibodies.

Serum autoantibodies in pristane induced lupus are regulated by neutrophil gelatinase associated lipocalin.

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.

Toll-like receptor 7-stimulated tumor necrosis factor α causes bone marrow damage in systemic lupus erythematosus.

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/ß/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.

Pleiotropic IFN-dependent and -independent effects of IRF5 on the pathogenesis of experimental lupus.

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 Ly6C(hi) 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.

Lupus-like disease and high interferon levels corresponding to trisomy of the type I interferon cluster on chromosome 9p.

OBJECTIVE: Systemic lupus erythematosus (SLE) is associated with type I interferons (IFNs) and can be induced by IFNalpha treatment. This study looked for evidence of autoimmunity in a pedigree consisting of 4 family members with a balanced translocation 9;21 and 2 members with an unbalanced translocation resulting in trisomy of the short (p) arm and part of the long (q) arm of chromosome 9. These latter 2 subjects had 3 copies of the IFN gene cluster. METHODS: Subjects were evaluated clinically and serologically for autoimmune disease. Expression levels of IFNalpha4, IFNbeta, the type I IFN-inducible gene Mx1, the type I IFN receptor, interleukin-6, and tumor necrosis factor alpha were determined by real-time polymerase chain reaction. Circulating plasmacytoid dendritic cells, the main IFN-producing cells, were quantified by flow cytometry. RESULTS: Both subjects with trisomy of chromosome 9p had a lupus-like syndrome with joint manifestations and antinuclear antibodies: one had anti-RNP and antiphospholipid autoantibodies, and the other had anti-Ro 60. The 3 family members with a balanced translocation 9;21 had no clinical or serologic evidence of autoimmunity, similar to that in relatives who were unaffected by the chromosomal translocation. In the 2 subjects with trisomy of 9p, high levels of IFNalpha/beta (comparable with those found in patients with SLE), increased signaling through the IFN receptor (as indicated by high Mx1 expression), and low levels of circulating plasmacytoid dendritic cells (as observed in patients with SLE) were evident. These abnormalities were not seen in individuals with a balanced translocation. CONCLUSION: Trisomy of the type I IFN cluster of chromosome 9p was associated with lupus-like autoimmunity and increased IFNalpha/beta and IFN receptor signaling. The data support the idea that abnormal regulation of type I IFN production is involved in the pathogenesis of SLE.

Type I interferon production by tertiary lymphoid tissue developing in response to 2,6,10,14-tetramethyl-pentadecane (pristane).

Lymphoid neogenesis is associated with antibody-mediated autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. Although systemic lupus erythematosus is the prototypical B-cell-mediated autoimmune disease, the role of lymphoid neogenesis in its pathogenesis is unknown. Intraperitoneal injection of 2,6,10,14-tetramethyl-pentadecane (TMPD, pristane) or mineral oil causes lipogranuloma formation in mice, but only TMPD-treated mice develop lupus. We report that lipogranulomas are a form of lymphoid neogenesis. Immunoperoxidase staining of lipogranulomas revealed B cells, CD4(+) T cells, and dendritic cells and in some cases organization into T- and B-cell zones. Lipogranulomas also expressed the lymphoid chemokines CCL21, CCL19, CXCL13, CXCL12, and CCL22. Expression of the type I interferon (IFN-I)-inducible genes Mx1, IRF7, IP-10, and ISG-15 was greatly increased in TMPD- versus mineral oil-induced lipogranulomas. Dendritic cells from TMPD lipogranulomas underwent activation/maturation with high CD86 and interleukin-12 expression. Magnetic bead depletion of dendritic cells markedly diminished IFN-inducible gene (Mx1) expression. We conclude that TMPD-induced lupus is associated with the formation of ectopic lymphoid tissue containing activated dendritic cells producing IFN-I and interleukin-12. In view of the increased IFN-I production in systemic lupus erythematosus, these studies suggest that IFN-I from ectopic lymphoid tissue could play a role in the pathogenesis of experimental lupus in mice.