Carla M. Cuda

Nonclassical Ly6C(-) Monocytes Drive the Development of Inflammatory Arthritis in Mice

Different subsets and/or polarized phenotypes of monocytes and macrophages may play distinct roles during the development and resolution of inflammation. Here, we demonstrate in a murine model of rheumatoid arthritis that nonclassical Ly6C(-) monocytes are required for the initiation and progression of sterile joint inflammation. Moreover, nonclassical Ly6C(-) monocytes differentiate into inflammatory macrophages (M1), which drive disease pathogenesis and display plasticity during the resolution phase. During the development of arthritis, these cells polarize toward an alternatively activated phenotype (M2), promoting the resolution of joint inflammation. The influx of Ly6C(-) monocytes and their subsequent classical and then alternative activation occurs without changes in synovial tissue-resident macrophages, which express markers of M2 polarization throughout the course of the arthritis and attenuate joint inflammation during the initiation phase. These data suggest that circulating Ly6C(-) monocytes recruited to the joint upon injury orchestrate the development and resolution of autoimmune joint inflammation.

Genetic Determination of T Cell Help in Loss of Tolerance to Nuclear Antigens

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.

NADPH Oxidase Deficiency Regulates Th Lineage Commitment and Modulates Autoimmunity

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.

Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span.

Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion.

Murine Lupus Susceptibility Locus Sle1a Controls Regulatory T Cell Number and Function through Multiple Mechanisms

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.

The NZM2410-derived lupus susceptibility locus Sle2c1 increases Th17 polarization and induces nephritis in fas-deficient mice

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.

Caspase-8 acts as a molecular rheostat to limit RIPK1- and MyD88-mediated dendritic cell activation

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.

Murine lupus susceptibility locus Sle1a requires the expression of two sub-loci to induce inflammatory T cells

The NZM2410-derived Sle1a lupus susceptibility locus induces activated autoreactive CD4+ T cells and reduces the number and function of Foxp3+ regulatory T cells (Tregs). In this study, we first showed that Sle1a contributes to autoimmunity by increasing antinuclear antibody production when expressed on either NZB or NZW heterozygous genomes, and by enhancing the chronic graft versus host disease response indicating an expansion of the autoreactive B-cell pool. Screening two non-overlapping recombinants, the Sle1a.1 and Sle1a.2 intervals that cover the entire Sle1a locus, revealed that both Sle1a.1 and Sle1a.2 were necessary for the full Sle1a phenotype. Sle1a.1, and to a lesser extent Sle1a.2, significantly affected CD4+ T-cell activation as well as Treg differentiation and function. Sle1a.2 also increased the production of autoreactive B cells. As the Sle1a.1 and Sle1a.2 intervals contain only 1 and 15 known genes, respectively, this study considerably reduces the number of candidate genes responsible for the production of autoreactive T cells. These results also show that the Sle1 locus is an excellent model for the genetic architecture of lupus, in which a major obligate phenotype results from the coexpression of multiple genetic variants with individual weak effects.

Cyclin-dependent kinase inhibitor p21, via its C-terminal domain, is essential for resolution of murine inflammatory arthritis.

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.

Defective response of CD4+ T cells to retinoic acid and TGFβ in systemic lupus erythematosus

IntroductionCD25+ 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.MethodsPeripheral 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.ResultsThe 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.ConclusionsWe 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.