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Background The risk for developing cardiovascular disease is greater in patients with rheumatoid arthritis (RA) than in the general population. While patients with RA also have dyslipidemia, the impact of dyslipidemia on the severity of inflammatory arthritis and associated cardiovascular disease is unclear. Currently, there are conflicting results regarding arthritis incidence in apolipoprotein E (ApoE) deficient mice, which spontaneously exhibit both hyperlipidemia and atherosclerosis. Here, we utilize a distinct approach to investigate the contribution of a hyperlipidemic environment on the development of arthritis and atherosclerosis in mice lacking ApoE. Methods K/BxN serum transfer-induced arthritis (STIA) was assessed in C57BL/6 (control) and ApoE−/− mice using clinical indices and immunohistochemical staining. Ankle synoviums were processed for flow cytometry. Aortic atherosclerosis was quantitated using Sudan IV staining. Serum cholesterol and cytokine levels were determined via enzymatic and luminex bead-based assays, respectively. Results ApoE−/− mice developed a sustained and enhanced semi-chronic inflammatory arthritis as compared to control mice. ApoE−/− mice had increased numbers of foamy macrophages, enhanced joint inflammation and amplified collagen deposition versus controls. The presence of arthritis did not exacerbate serum cholesterol levels or significantly augment the level of atherosclerosis in ApoE−/− mice. However, arthritic ApoE−/− mice exhibited a marked elevation of IL-6 as compared to non-arthritic ApoE−/− mice and arthritic C57BL/6 mice. Conclusions Loss of ApoE potentiates a semi-chronic inflammatory arthritis. This heightened inflammatory response was associated with an increase in circulating IL-6 and in the number of foamy macrophages within the joint. Moreover, the foamy macrophages within the arthritic joint are reminiscent of those within unstable atherosclerotic lesions and suggest a pathologic role for foamy macrophages in propagating arthritis. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0912-y) contains supplementary material, which is available to authorized users.

Autoimmunity occurs through a break in tolerance, which is considered to be mediated by a failure to delete autoreactive immune cells. The central mechanism for deleting cells is controlled by the apoptotic machinery. Apoptosis occurs via two distinct pathways, an extrinsic pathway involving transduction of an apoptotic signal following aggregation of a death receptor, such as Fas, to its ligand, Fas ligand (FasL), and an intrinsic pathway that signals through the mitochondria and is regulated by the bcl-2 family. In the extrinsic pathway, binding of homotrimeric FasL to Fas facilitates recruitment of both Fas-associated death domain protein (FADD) and pro-caspase-8 leading to the activation of caspase-8 and subsequent degradative phase of apoptosis (1). This process may be inhibited by cellular FADD-like IL-1β-converting enzyme (FLICE)-inhibitory protein (cFLIP), which is also recruited by FADD and acts as an endogenous suppressor of the Fas pathway (1). Mice mutant for Fas (lpr) mice show normal postnatal development, but over time exhibit splenomegaly, lymphadenopathy, hypergammaglobulinemia, autoantibodies, glomerulonephritis, and the presence of double negative T-cells (CD3+CD4-CD8-B220+), with disease severity highly dependent upon strain background (2). The advent of conditional deletion of genes has yielded novel, unexpected, and controversial results regarding specific effects of loss of Fas within the various hematopoietic cell populations. While previous studies have extensively examined the role that Fas plays in lymphocytes and dendritic cells (3-5), few or no studies have directly examined the cell-intrinsic role of Fas in myeloid cells and its contribution to autoimmunity. Here, mice were generated that conditionally lack Fas only in the myeloid cell compartment (CreLysMFasflox/flox). These mice displayed a disruption in monocyte/macrophage and granulocyte homeostasis that led to altered activation of dendritic cells and lymphocytes in a paracrine fashion. Moreover, CreLysMFasflox/flox mice presented with splenomegaly, as well as autoantibodies and proinflammatory cytokines/chemokines. Furthermore, deposited immune complexes in kidneys and glomerulonephritis were observed in mice with myeloid cell-specific loss of Fas. Taken together, these data indicate that loss of Fas in myeloid cells is sufficient to induce an inflammatory phenotype in mice reminiscent of SLE-like disease, suggesting a role of specifically targeting Fas in its treatment.

Objective Sle2 is a lupus susceptibility locus that has been linked to glomerulonephritis in the NZM2410 mouse. By itself, Sle2 does not induce any autoimmune pathology but results in the accumulation of B-1a cells. This study was designed to assess the contribution of Sle2 to the pathogenesis of autoimmunity. Methods Sle2 or its subcongenic intervals (Sle2a, Sle2b, and Sle2c1) were bred to Fas-deficient B6.lpr mice. Lymphoid phenotypes, which were focused on T cells, were assessed by flow cytometry, and histopathologic changes were compared between cohorts of B6.Sle2.lpr congenic mice and B6.lpr mice of ages up to 6 months. Results Sle2 synergized with lpr, resulting in a greatly accelerated lymphadenopathy that largely targeted T cells and mapped to the Sle2c1 locus. This locus has been identified as the main contributor to B-1a cell expansion. Further analyses showed that Sle2c1 expression skewed the differentiation and polarization of Fas-deficient T cells, with a reduction of the CD4+CD25+FoxP3+ regulatory T cell subset and an expansion of the Th17 cells. This was associated with a high number of T cell infiltrates that promoted severe nephritis and dermatitis in the B6.Sle2c1.lpr mice. Conclusion These results show that Sle2c1 contributes to lupus pathogenesis by affecting T cell differentiation in combination with other susceptibility loci, such as lpr. The significance of the cosegregation of this phenotype and B-1a cell expansion in Sle2c1-expressing mice in relation to the pathogenesis of lupus is discussed.

The Sle1 locus is a key determinant of lupus susceptibility in the NZM2410 mouse model. Within Sle1, we have previously shown that Sle1a expression enhances activation levels and effector functions of CD4+ T cells and reduces the size of the CD4+CD25+Foxp3+ regulatory T cell subset, leading to the production of autoreactive T cells that provide help to chromatin-specific B cells. In this study, we show that Sle1a CD4+ T cells express high levels of ICOS, which is consistent with their increased ability to help autoreactive B cells. Furthermore, Sle1a CD4+CD25+ T cells express low levels of Foxp3. Mixed bone marrow chimeras demonstrated that these phenotypes require Sle1a to be expressed in the affected CD4+ T cells. Expression of other markers generally associated with regulatory T cells (Tregs) was similar regardless of Sle1a expression in Foxp3+ cells. This result, along with in vitro and in vivo suppression studies, suggests that Sle1a controls the number of Tregs rather than their function on a per cell basis. Both in vitro and in vivo suppression assays also showed that Sle1a expression induced effector T cells to be resistant to Treg suppression, as well as dendritic cells to overproduce IL-6, which inhibits Treg suppression. Overall, these results show that Sle1a controls both Treg number and function by multiple mechanisms, directly on the Tregs themselves and indirectly through the response of effector T cells and the regulatory role of dendritic cells.

Introduction Although caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death. As cells of the innate immune system, and in particular macrophages, are now at the forefront of autoimmune disease pathogenesis, we examined the potential involvement of caspase-8 within this population. Methods CreLysMCasp8fl/fl mice were bred via a cross between Casp8fl/fl mice and CreLysM mice, and RIPK3−/−CreLysMCasp8fl/fl mice were generated to assess the contribution of receptor-interacting serine-threonine kinase (RIPK)3. Immunohistochemical and immunofluorescence analyses were used to examine renal damage. Flow cytometric analysis was employed to characterize splenocyte distribution and activation. CreLysMCasp8fl/fl mice were treated with either Toll-like receptor (TLR) agonists or oral antibiotics to assess their response to TLR activation or TLR agonist removal. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure cytokine/chemokine and immunoglobulin levels in serum and cytokine levels in cell culture studies. In vitro cell culture was used to assess macrophage response to cell death stimuli, TLR activation, and M1/M2 polarization. Data were compared using the Mann–Whitney U test. Results Loss of caspase-8 expression in macrophages promotes onset of a mild systemic inflammatory disease, which is preventable by the deletion of RIPK3. In vitro cell culture studies reveal that caspase-8–deficient macrophages are prone to a caspase-independent death in response to death receptor ligation; yet, caspase-8–deficient macrophages are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations. Loss of caspase-8 in macrophages dictates the response to TLR activation, as injection of TLR ligands upregulates expression of costimulatory CD86 on the Ly6ChighCD11b+F4/80+ splenic cells, and oral antibiotic treatment to remove microbiota prevents splenomegaly and lymphadenopathy in CreLysMCasp8fl/fl mice. Further, caspase-8–deficient macrophages are hyperresponsive to TLR activation and exhibit aberrant M1 macrophage polarization due to RIPK activity. Conclusions These data demonstrate that caspase-8 functions uniquely in macrophages by controlling the response to TLR activation and macrophage polarization in an RIPK-dependent manner. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0794-z) contains supplementary material, which is available to authorized users.

Cortical dysplasia is frequently associated with epilepsy but mechanisms underlying this association are poorly understood. Rats irradiated in utero serve as an injury-based model of cortical dysplasia. Prior studies in mature rats have shown a selective reduction in the number of neocortical interneurons after in utero irradiation. This study attempted to clarify the nature of the radiation injury to the developing neocortical GABAergic system after exposure to gamma-irradiation on the 17th day of gestation (E17). Stereological methods were used to quantify absolute numbers of total neurons (TN) and GABAergic neurons in the neocortex on E21 and postnatal day 6 (P6). In irradiated rats, TN was decreased to about 50% of controls at both time points. However, TN doubled between the 2 time points, even in irradiated animals. In controls, GABAergic neurons increased 10-fold between E21 and P6, but there was no difference in GABAergic counts between the 2 time points in irradiated animals. This led to a dramatic reduction in the percentage of neocortical neurons that were GABAergic in irradiated animals at P6 (9% vs 18%). This study shows that, in contrast to non-GABAergic neurons, the neocortical GABAergic system has a limited capacity to recover from radiation-induced in utero injury.

Caspase-8, an executioner enzyme in the death receptor pathway, was shown to initiate apoptosis and suppress necroptosis. In this study, we identify a novel, cell death–independent role for caspase-8 in dendritic cells (DCs): DC-specific expression of caspase-8 prevents the onset of systemic autoimmunity. Failure to express caspase-8 has no effect on the lifespan of DCs but instead leads to an enhanced intrinsic activation and, subsequently, more mature and autoreactive lymphocytes. Uncontrolled TLR activation in a RIPK1-dependent manner is responsible for the enhanced functionality of caspase-8–deficient DCs, because deletion of the TLR-signaling mediator, MyD88, ameliorates systemic autoimmunity induced by caspase-8 deficiency. Taken together, these data demonstrate that caspase-8 functions in a cell type–specific manner and acts uniquely in DCs to maintain tolerance.

Polymorphisms in the transcription factor interferon regulatory factor 5 (IRF5) are associated with an increased risk of developing rheumatoid arthritis (RA). This study was undertaken to determine the role of IRF5 in a mouse model of arthritis development.K/BxN serum-transfer arthritis was induced in mice deficient in IRF5, or lacking IRF5 only in myeloid cells, and arthritis severity was evaluated. K/BxN arthritis was also induced in mice deficient in TRIF, Toll-like receptor 2 (TLR2), TLR3, TLR4, and TLR7 to determine the pathways through which IRF5 might promote arthritis. In vitro studies were performed to determine the role of IRF5 in interleukin-1 (IL-1) receptor and TLR signaling.Arthritis severity was reduced in IRF5-deficient, TRIF-deficient, TLR3-deficient, and TLR7-deficient mice. The expression of multiple genes regulating neutrophil recruitment or function and bioactive IL-1β formation was reduced in the joints during active arthritis in IRF5-deficient mice. In vitro studies showed that TLR7 and the TRIF-dependent TLR3 pathway induce proinflammatory cytokine production in disease-relevant cell types in an IRF5-dependent manner.Our findings indicate that IRF5 contributes to disease pathogenesis in inflammatory arthritis. This is likely due at least in part to the role of IRF5 in mediating proinflammatory cytokine production downstream of TLR7 and TLR3. Since TLR7 and TLR3 are both RNA-sensing TLRs, this suggests that endogenous RNA ligands present in the inflamed joint promote arthritis development. These findings may be relevant to human RA, since RNA capable of activating TLR7 and TLR3 is present in synovial fluid and TLR7 and TLR3 are up-regulated in the joints of RA patients.

The innate immune system responds to infection and tissue damage by activating cytosolic sensory complexes called 'inflammasomes'. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the inflammasome adaptor ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but the regulation of ALR inflammasomes is poorly understood. Here we identified the PYRIN domain (PYD)-only protein POP3, which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus-induced activation of ALR inflammasomes in vivo. Data obtained with a mouse model with macrophage-specific POP3 expression emphasize the importance of the regulation of ALR inflammasomes in monocytes and macrophages.

A nonapoptotic role of Fas signaling has been implicated in the regulation of inflammation and innate immunity. This study was undertaken to elucidate the contribution of Fas signaling in macrophages to the development of arthritis.K/BxN serum-transfer arthritis was induced in a mouse line in which Fas was conditionally deleted in the myeloid lineage (Cre(LysM) Fas(flox/flox) mice). The arthritis was assessed clinically and histologically. Expression of interleukin-1β (IL-1β), CXCL5, IL-10, IL-6, and gp96 was determined by enzyme-linked immunosorbent assay. Bone marrow-derived macrophages were activated with IL-1β and gp96. Cell phenotype and apoptosis were analyzed by flow cytometry.Arthritis onset in Cre(LysM) Fas(flox/flox) mice was comparable with that observed in control mice; however, resolution was accelerated during the chronic phase. The attenuated arthritis was associated with reduced articular expression of the endogenous Toll-like receptor 2 (TLR-2) ligand gp96 and the neutrophil chemotactic chemokine CXCL5, and enhanced expression of IL-10. Activation with IL-1β or gp96 induced increased IL-10 expression in Fas-deficient murine macrophages compared with control macrophages. IL-10 suppressed IL-6 and CXCL5 expression induced by IL-1β plus gp96. IL-1β-mediated activation of ERK, which regulates IL-10 expression, was increased in Fas-deficient mouse macrophages.Taken together, our findings indicate that impaired Fas signaling results in enhanced expression of antiinflammatory IL-10 and reduced expression of gp96, and these effects are associated with accelerated resolution of inflammation during the chronic phase of arthritis. These observations suggest that strategies to reduce endogenous TLR ligands and increase IL-10 may be beneficial in the treatment of rheumatoid arthritis.

The death receptor Fas is a critical mediator of the extrinsic apoptotic pathway, and its role in mediating lymphoproliferation has been extensively examined. The present study was undertaken to investigate the impact of myeloid cell-specific loss of Fas.Mice with Fas flanked by loxP sites (Fas(flox/flox) ) were crossed with mice expressing Cre under control of the murine lysozyme M gene promoter (Cre(LysM) ), which functions in mature lysozyme-expressing cells of the myelomonocytic lineage. The genotype for Cre(LysM) Fas(flox/flox) mice was verified by polymerase chain reaction and flow cytometric analysis. Flow cytometric analysis was also used to characterize myeloid, dendritic, and lymphoid cell distribution and activation in bone marrow, blood, and spleen. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure serum cytokine/chemokine and immunoglobulin levels. Renal damage or dysfunction was examined by immunohistochemical and immunofluorescence analysis.Cre(LysM) Fas(flox/flox) mice exhibited a systemic lupus erythematosus (SLE)-like disease that included leukocytosis, splenomegaly, hypergammaglobulinemia, antinuclear autoantibody and proinflammatory cytokine production, and glomerulonephritis. Loss of Fas in myeloid cells increased levels of both Gr-1(low) and Gr-1(intermediate) blood monocytes and splenic macrophages and, in a paracrine manner, incited activation of conventional dendritic cells and lymphocytes in Cre(LysM) Fas(flox/flox) mice.Taken together, these results suggest that loss of Fas in myeloid cells is sufficient to induce inflammatory phenotypes in mice, reminiscent of an SLE-like disease. Thus, Fas in myeloid cells may be considered a suppressor of systemic autoimmunity.

Sle1a.1 is part of the Sle1 susceptibility locus, which has the strongest association with lupus nephritis in the NZM2410 mouse model. In this study, we show that Sle1a.1 results in the production of activated and autoreactive CD4+ T cells. Additionally, Sle1a.1 expression reduces the peripheral regulatory T cell pool, as well as induces a defective response of CD4+ T cells to the retinoic acid expansion of TGF-β–induced regulatory T cells. At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d overexpression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells and to decrease their apoptotic response to retinoic acid. PBX1-d is expressed more frequently in the CD4+ T cells from lupus patients than from healthy controls, and its presence correlates with an increased central memory T cell population. These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance.

OBJECTIVE: The mechanism responsible for persistent synovial inflammation in rheumatoid arthritis (RA) is unknown. Previously, we demonstrated that expression of the cyclin-dependent kinase inhibitor p21 is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. The present study was undertaken to investigate the role and mechanism of p21-mediated suppression of experimental inflammatory arthritis. METHODS: Experimental arthritis was induced in wild-type or p21-/- (C57BL/6) mice, using the K/BxN serum-transfer model. Mice were administered p21 peptide mimetics as a prophylactic for arthritis development. Lipopolysaccharide-induced cytokine and signal transduction pathways in macrophages that were treated with p21 peptide mimetics were examined by Luminex-based assay, flow cytometry, or enzyme-linked immunosorbent assay. RESULTS: Enhanced and sustained development of experimental inflammatory arthritis, associated with markedly increased numbers of macrophages and severe articular destruction, was observed in p21-/- mice. Administration of a p21 peptide mimetic suppressed activation of macrophages and reduced the severity of experimental arthritis in p21-intact mice only. Mechanistically, treatment with the p21 peptide mimetic led to activation of the serine/threonine kinase Akt and subsequent reduction of the activated isoform of p38 MAPK in macrophages. CONCLUSION: These are the first reported data to reveal that p21 has a key role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA., The authors are grateful for the assistance of staff of the Washington University School of Medicine Center for Musculoskeletal Biology and Medicine and the Northwestern University Feinberg School of Medicine Cell Imaging Core Facility and the Methodology and Data Management Core.

Reactive oxygen species are used by the immune system to eliminate infections; however, they may also serve as signaling intermediates to coordinate the efforts of the innate and adaptive immune systems. In this study, we show that by eliminating macrophage and T cell superoxide production through the NADPH oxidase (NOX), T cell polarization was altered. After stimulation with immobilized anti-CD3 and anti-CD28 or priming recall, T cells from NOX-deficient mice exhibited a skewed Th17 phenotype, whereas NOX-intact cells produced cytokines indicative of a Th1 response. These findings were corroborated in vivo by studying two different autoimmune diseases mediated by Th17 or Th1 pathogenic T cell responses. NOX-deficient NOD mice were Th17 prone with a concomitant susceptibility to experimental allergic encephalomyelitis and significant protection against type 1 diabetes. These data validate the role of superoxide in shaping Th responses and as a signaling intermediate to modulate Th17 and Th1 T cell responses.

Sle1 is a major lupus susceptibility locus in NZM2410 lupus model that is associated with a loss of tolerance to nuclear Ags. At least three genes, Sle1a, Sle1b, and Sle1c contribute to Sle1, and their relative role in lupus pathogenesis is unknown. We show here that Sle1-expressing CD4+ T cells present an activated phenotype associated with increased proliferation and cytokine production. In addition, Sle1 CD4+ T cells provide help to anti-chromatin B cells to produce anti-nuclear antibodies, whether or not these B cells express Sle1. The Sle1a locus alone accounts for all these Sle1 phenotypes, implying that a specific genetic defect in Sle1a is necessary and sufficient to produce autoreactive T cells. However, Sle1c induces intermediate T cell activation and only provides help to Sle1-expressing anti-chromatin-producing B cells, demonstrating the synergic interactions between Sle1c T and Sle1 B cells. Moreover, Sle1a and Sle1c were associated with a significantly reduced level of CD4+CD25+ regulatory T cells that precedes autoantibody production, suggesting a causal relationship with the generation of autoreactive T cells. Our study identifies for the first time that a specific genetic defect is responsible for lupus pathogenesis by inducing autoreactive T cells to break self-tolerance and that this genetic defect is also associated with a decreased number of regulatory T cells.

CD25(+) FOXP3(+) CD4(+) regulatory T cells (Tregs) are induced by transforming growth factor β (TGFβ) and further expanded by retinoic acid (RA). We have previously shown that this process was defective in T cells from lupus-prone mice expressing the novel isoform of the Pbx1 gene, Pbx1-d. This study tested the hypothesis that CD4(+) T cells from systemic lupus erythematosus (SLE) patients exhibited similar defects in Treg induction in response to TGFβ and RA, and that PBX1-d expression is associated with this defect.Peripheral blood mononuclear cells (PBMCs) were collected from 142 SLE patients and 83 healthy controls (HCs). The frequency of total, memory and naïve CD4(+) T cells was measured by flow cytometry on fresh cells. PBX1 isoform expression in purified CD4(+) T cells was determined by reverse transcription polymerase chain reaction (RT-PCR). PBMCs were stimulated for three days with anti-CD3 and anti-CD28 in the presence or absence of TGFβ and RA. The expression of CD25 and FOXP3 on CD4(+) T cells was then determined by flow cytometry. In vitro suppression assays were performed with sorted CD25(+) and CD25(-) FOXP3(+) T cells. CD4(+) T cell subsets or their expansion were compared between patients and HCs with two-tailed Mann-Whitney tests and correlations between the frequencies of two subsets were tested with Spearman tests.The percentage of CD25(-) FOXP3(+) CD4(+) (CD25(-) Tregs) T cells was greater in SLE patients than in HCs, but these cells, contrary to their matched CD25(+) counterparts, did not show a suppressive activity. RA-expansion of TGFβ-induced CD25(+) Tregs was significantly lower in SLE patients than in HCs, although SLE Tregs expanded significantly more than HCs in response to either RA or TGFβ alone. Defective responses were also observed for the SLE CD25(-) Tregs and CD25(+) FOXP3(-) activated CD4(+) T cells as compared to controls. PBX1-d expression did not affect Treg induction, but it significantly reduced the expansion of CD25- Tregs and prevented the reduction of the activated CD25(+) FOXP3(-) CD4(+) T cell subset by the combination of TGFβ and RA.We demonstrated that the induction of Tregs by TGFβ and RA was defective in SLE patients and that PBX1-d expression in CD4(+) T cells is associated with an impaired regulation of FOXP3 and CD25 by TGFβ and RA on these cells. These results suggest an impaired integration of the TGFβ and RA signals in SLE T cells and implicate the PBX1 gene in this process.